The prevalence of Mycobacterium abscessus (MABS) infections in people with cystic fibrosis (pwCF) is increasing. Macrophages are key phagocytic cells that recognize bacteria via cell surface receptors, engulf them into phagosomes, and then utilize diverse killing strategies. Here, we used human primary monocyte-derived macrophages (MDMs) from healthy controls (HCs) and pwCF to investigate how they processed and killed MABS. Expression of phagocytosis-related pattern recognition receptors (TLR2, Dectin-1, Dectin-2, and MARCO), engulfment of MABS, and lysosomal acidity were all reduced in CF-MDMs. MABS-infected CF-MDMs also had reduced mitochondrial mass, mitochondrial reactive oxygen species (mitoROS) production, relative intracellular zinc levels and inducible mRNA expression of the antibacterial zinc transporters, SLC30A1 and SLC39A8. Stimulation of mitoROS production in HC-MDMs with antimycin A reduced intracellular loads of MABS, confirming that MABS are sensitive to this mechanism of killing and suggesting that the mitoROS defect in MABS-infected CF-MDMs compromises bacterial killing. Accordingly, CF-MDMs failed to control MABS infection, with these cells allowing significantly increased intracellular MABS survival and expansion over 6 d. While treatment with the CFTR modulator, elexacaftor-tezacaftor-ivacaftor (ETI) did increase CFTR channel function and corrected CF macrophage functions to some degree, this was not sufficient to increase MABS killing. Taken together, our findings suggest important roles for functional CFTR in internalization and killing of MABS within macrophages, with CFTR dysfunction supporting MABS survival and replication in macrophages. Under our experimental conditions, ETI treatment failed to fully restore macrophage functions against MABS, highlighting the need for alternative, host-targeted approaches for improving macrophage functions in CF.

Introduction The Australian Guidelines for the Assessment and Diagnosis of Fetal Alcohol Spectrum Disorder were released in May 2025. These guidelines, updated from the 2016 Australian Guide, were developed through extensive stakeholder consultation, a comprehensive review of empirical evidence and the application of a novel Grading of Recommendations, Development and Evaluation (GRADE)-based approach for developing evidence-based diagnostic criteria.Recommendations The guidelines include 11 GRADE-based recommendations, forming the core of the diagnostic criteria, and 11 lived experience statements, supporting client-centred assessment approaches. The guidelines also feature 40 good practice statements and 18 implementation considerations, tools and tips as practical resources and supports for practitioners.Changes in Management as a Result of the Guidelines Key changes in the new guidelines include: A minimum prenatal alcohol exposure threshold for diagnosis to support more precise identification of fetal alcohol spectrum disorder (FASD); The assessment of neurodevelopmental impairments and functioning has been refined, including detailed advice for practitioners seeking to identify clinically significant impairments; Inclusion/exclusion of neurodevelopmental domains as part of the assessment; and Revisions to some of the content of the included neurodevelopmental domains. Changes have also been made with attention to the needs of First Nations Australians and people from other culturally and linguistically diverse backgrounds. The guidelines advocate for the use of shared decision-making approaches, promoting collaboration between practitioners, individuals attending for assessment and their support network. Importantly, the guidelines will enable an accessible, holistic and flexible assessment process, supporting timely and accurate assessment and diagnosis of FASD in Australia. The rigorous development process also provides practitioners with confidence in the guidelines, promoting increased uptake of assessment and diagnostic practices, and informing international diagnostic criteria and guidelines.

BACKGROUND:Fetal alcohol spectrum disorder (FASD) is associated with a range of developmental, behavioral, and physical impairments. While much research to date has focused on developmental and behavioral aspects of FASD, there is growing evidence that children with FASD may experience alterations in renal and cardiovascular function. By also comparing eating behaviors between children with FASD and typically developing children, this may provide some clues to the underlying causes of these physiological changes. METHODS:In this study, we recruited children and adolescents with (n = 34) and without (n = 22) a diagnosis of FASD. We measured several cardiovascular and renal outcomes and analyzed potential associations with eating behaviors related to food approach and avoidance, assessed from caregiver report using the Children's Eating Behavior Questionnaire (CEBQ). RESULTS:Children with FASD had significantly elevated urinary sodium-to-potassium (Na+/K+) ratio, a trend for increasing urine sodium (Na+), and a reduction in the normal age-associated rise in urine creatinine compared to typically developing controls. The FASD group also exhibited significantly elevated resting heart rate. Additionally, those with a diagnosis of FASD had higher scores for subscales of food approach (food responsiveness, emotional overeating, desire to drink) and a trend toward a higher food avoidance score on the CEBQ. A subset of these behavioral scores was associated with cardiovascular but not renal outcomes. CONCLUSIONS:Together, these findings suggest that renal and cardiovascular function may be altered in children and adolescents with FASD, which may be linked to abnormal food-related behaviors and/or other clinical or demographic factors. While the mechanisms underlying these early-life physiological changes are not fully understood, it is likely they contribute to poorer long-term health outcomes. Further research is needed to validate these preliminary findings in larger cohorts, explore the underlying mechanisms and investigate potential long-term implications of these findings.

INTRODUCTION:The Australian Guide to the Diagnosis of fetal alcohol spectrum disorder (FASD), developed in 2016, is currently under review. This study aimed to understand how the Guide is used in practice and identify factors influencing its implementation. METHODS:A cross-sectional online survey was conducted with Australian health practitioners involved in the assessment and diagnosis of FASD. The Clinician Guideline Determinants Questionnaire was used. Quantitative data were summarised descriptively and content analysis was applied to open-ended questions. RESULTS:Of 333 survey sessions, 232 participants consented and 136 completed the survey. Most respondents were paediatricians (n = 54, 37%) or psychologists (n = 49, 33%). Although most clinicians felt confident using the Guide, only 44.1% agreed it clearly described the underlying evidence, and just 21% felt it adequately reflected patient preferences. Key enablers included clear, easy-to-follow guidance (n = 45, 36.3%); ease of access (n = 18, 14.5%) and familiarity with the Guide (n = 14, 11.3%). Common barriers were lack of access to appropriate clinicians, time and cost constraints (n = 25, 24.8%); concerns about the evidence base (n = 16, 15.8%); and lack of familiarity or skills (n = 8, 7.9%). DISCUSSION AND CONCLUSIONS:Findings highlight strong clinician engagement with the 2016 Guide but also identify areas for improvement, particularly around evidence transparency and alignment with person-centred care. By engaging with stakeholders and improving guidelines based on clinician feedback, we can improve the quality of care and support for individuals with FASD and their families.

Fetal alcohol spectrum disorder (FASD) is a significant public health concern, yet there is no internationally agreed set of diagnostic criteria or summary of underlying evidence to inform diagnostic decision-making. This systematic review assesses associations of prenatal alcohol exposure (PAE) and outcomes of diagnostic assessments, providing an evidence base for the improvement of FASD diagnostic criteria. Six databases were searched (inception–February 2023). Case-controls or cohort studies examining associations between participants with/without PAE or a FASD diagnosis and the domains of physical size, dysmorphology, functional neurodevelopment and/or brain structure/neurology were included. Excluded studies were non-empirical, sample size < 10, PAE determined via biological markers only, or no suitable comparison group. Summary data were extracted and associations between outcomes and standardised levels of PAE or FASD diagnosis determined using random-effects meta-analyses. Certainty of the evidence was assessed using GRADE. Of the 306 included studies, 106 reported physical size, 43 dysmorphology, 195 functional neurodevelopment and 110 structural/neurological outcomes, with 292 different outcomes examined. There was a dose–response relationship between PAE and head circumference, as well as measures of physical size, particularly at birth. There was also an association between higher PAE levels and characteristic sentinel facial dysmorphology, as well as many of the current functional neurodevelopmental outcomes considered during diagnosis. However, data were often lacking across the full range of exposures. There was a lack of evidence from studies examining PAE to support inclusion of non-sentinel dysmorphic features, social cognition, speech-sound impairments, neurological conditions, seizures, sensory processing or structural brain abnormalities (via clinical MRI) in diagnostic criteria. GRADE ratings ranged from very low to moderate certainty of evidence. This comprehensive review provides guidance on which components are most useful to consider in the diagnostic criteria for FASD. It also highlights numerous gaps in the available evidence. Future well-designed pregnancy cohort studies should specifically focus on dose–response relationships between PAE and dysmorphology, neurodevelopment and brain structure/neurological outcomes. PROSPERO: CRD42021230522.

Children with neurodevelopmental disorders are currently overrepresented in the out-of-home-care (OOHC) system. This overrepresentation has created an increasing need for staff and carers to be equipped to handle the complexities these conditions entail. The aim of the present study was to gather information to understand the current service needs, practices, and knowledge within a private provider of OOHC services in Queensland. This included identifying barriers to the implementation of the Australian National Clinical Assessment Framework, which aims to facilitate appropriate screening, assessment and intervention provision for children and young people in OOHC. Twenty-five staff and 15 caregivers participated in an online survey. Results demonstrated that both staff and carers felt they needed additional training in understanding and supporting children with neu-rodevelopmental disorders and identified a number of barriers to accessing services. Barriers included: insuffi-cient access to information (e.g., lack of training, health records missing or incomplete and lack of appropriate pre-placement consultation) and lack of available resources (e.g., extensive wait times for services due to reliance on public health system and 'red tape' systemic issues). The results also highlighted the level of unmet physical, developmental and psychological needs for children and young people in care, with a third of carers reporting concerns. Overall, the current study provides critical recommendations for future implementation strategies and interventions to better meet the needs of children and young people in the Queensland OOHC system.

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30–200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein–Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.

Exosomes are nano-vesicles (30–150 nm) which may be useful as therapeutic delivery vehicles and as diagnostic biomarkers. Exosomes are produced naturally within the human body and therefore are not prone to immunogenicity effects which would otherwise destroy unelicited foreign bodies. Clinically, they have been regarded as ideal candidates for applications relating to biomarker developments for the early detection of different diseases. Furthermore, exosomes may be of interest as potential drug delivery vehicles, which may improve factors such as bioavailability of loaded molecular cargo, side effect profiles, off-target effects, and pharmacokinetics of drug molecules. In this review, the therapeutic potential of exosomes and their use as clinical biomarkers for early diagnostics will be explored, alongside exosomes as therapeutic delivery vehicles. This review will evaluate techniques for cargo loading, and the capacity of loaded exosomes to improve various reproductive disease states. It becomes important, therefore, to consider factors such as loading efficiency, loading methods, cell viability, exosomal sources, exosome isolation, and the potential therapeutic benefits of exosomes. Issues related to targeted drug delivery will also be discussed. Finally, the variety of therapeutic cargo and the application of appropriate loading methods is explored, in the context of establishing clinical utility. Graphical abstract Exosomes have more recently been widely accpeted as potential tools for disease diagnostics and the targeted delivery of certain therapeutic molecules–and in due time exosomes will be utilised more commonly within the clinical setting. Specifically, exosomal biomarkers can be identified and related to various detrimental conditions which occur during pregnancy. Considering, this review will explore the potential future of exosomes as both diagnostic tools and therapeutic delivery vehicles to treat related conditions, including the challenges which exist towards incorporating exosomes within the clinical environment to benefit patients.

Abnormal and dysregulated neuroinflammation has been linked to many neurological disorders and neurodegenerative diseases. Understanding the mechanisms of neuroinflammation, their impact on neurodevelopment and how neuroinflammation might be modulated, are currently considered to be critical to improving neurological treatment. ReNcell CX (originating from the cortical region) and VM (originating from the ventral mesencephalon) are human immortalised neural stem cell lines, that have the potential to be used as experimental models for investigating neuroinflammation in vitro. However, the information on the inflammation response of these cells is limited. This is especially more so for undifferentiated ReNcells. In this report we demonstrate using ELISA that cultured, undifferentiated ReNcell CX and VM produce significant amounts of IL-6 in response to IL-1β treatment, but not to LPS treatment. Additionally, conventional RT-PCR showed that ReNcell CX cells expressed TNFR1 and NF-κB, whereas ReNcell VM expressed only NF-κB. Our results encourage further investigation into the relationship between 1L-1β and IL-6 in both ReNcell CX and VM. Moreover, TNF-α treatment might potentially affect neuroinflammation in ReNcell CX, while activation of the NF-κB pathway could also play a critical part in neuroinflammation.

Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A ‘gold-standard’ method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

Abstract Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.

Fetal alcohol spectrum disorder (FASD) is a prevalent neurodevelopmental condition. Despite FASD being recognized as a clinical disorder there is no globally agreed set of diagnostic criteria. Accurate and timely diagnosis of FASD is imperative to inform clinical care, optimize outcomes for individuals accessing assessments and their families, as well as for research and prevention strategies. To inform movement towards a unified approach, the present study aimed to capture an international perspective on current FASD diagnostic criteria, as well as potential barriers and facilitators to unification. An online survey was created using REDCap and sent to clinics identified and contacted via internet searches. Quantitative data were presented using descriptive statistics and open-ended questions analysed using content analysis. The survey captured information about each clinic’s current diagnostic approach, whether they would support a unified method, and the barriers and facilitators for a consistent international FASD diagnostic approach. Fifty-five (37.4%) of 147 FASD clinics identified worldwide participated. The majority (n = 50, 90.9%) of respondents supported a unified approach. Content analysis identified a lack of collaboration as a key barrier, while strong leadership in guideline creation and implementation emerged as a central facilitator. These barriers and facilitators can be used to guide future collaborative efforts towards implementing consistent diagnostic criteria.

BACKGROUND:Exaggerated neutrophil-dominated inflammation underlies progressive cystic fibrosis (CF) lung disease. Older studies reported a defective respiratory burst in CF, but more recent studies suggest neutrophil function is normal. METHODS:We measured the amount and rate of reactive oxygen species (ROS) during PMA-stimulated respiratory burst activity in children [70 CF, 13 disease controls, 19 health controls] and adults [31 CF, 14 health controls] in neutrophils harvested from peripheral blood. Blood was collected from participants with CF when clinically stable (60 children, 9 adults) and on hospital admission (38 children, 24 adults) and discharge (18 children, 21 adults) for acute pulmonary exacerbations. RESULTS:When clinically stable, children with CF had lower ROS production [median 318,633, 25% 136,810 - 75% 569,523 RLU] than disease controls [median 599,459, 25% 425,566 - 75% 730,527 RLU] and healthy controls [median 534,073, 25% 334,057 - 75% 738,593 RLU] (p = 0.008). The rate of ROS production was also lower (p = 0.029). In neither children nor adults with CF did ROS production increase on hospital admission for acute pulmonary exacerbation, nor fall prior to discharge. There were no associations between ROS production and high-sensitivity C-reactive protein (indicating systemic inflammation) in either children or adults with CF. CONCLUSIONS:Our data do not support a role for exaggerated respiratory burst activity contributing to the exaggerated neutrophil-dominated inflammation seen with CF lung disease.

Proteomic analysis of exosomes (EX) poses a significant challenge. A ‘gold-standard’ method for plasma EX enrichment for downstream proteomic analysis is yet to be established. Our group has performed a comprehensive study of multi-dimensional enrichment methods to determine their efficiency for protein isolation. Methods were evaluated for their capacity to a) successfully isolate and enrich EX from blood plasma, b) minimise the presence of highly abundant plasma proteins, and c) result in the optimum representation of EX proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS). Blood plasma from four animals (Bos taurus) of similar physical attributes and genetics were used. Three methods of EX enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These enrichment methods were combined to create four groups for methodological evaluation: UC+SEC, UC+SEC+UF, SEC+UC and SEC+UF. UC+SEC yielded the highest number of protein IDs. Plasma protein identification was the least in SEC+UC, but this method yielded the lowest number of protein IDs overall. UC+SEC+UF decreased EX protein ID and did not improve purity compared to UC+SEC. Our data suggest that the method and sequence of EX enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

The present study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition.The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated using Information-Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison is made between their proteomic profiles.IDA analyses revealed similarities and differences in protein content. About 229 and 239 proteins were identified in the human and bovine milk exosome proteome, respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common in both groups. These included proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophilin, perilipin-2, and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes.The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance. A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, hypothetical proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to varying degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets.