Shivanthan Shanthikumar

A common technical artifact in droplet-based single-cell RNA sequencing (scRNA-seq) is the capture of two cells in the same droplet, termed doublets. Doublets may present as hybrid cell types, and their removal is a critical step during quality control. Another form of contamination is ambient RNA, which originates from free floating RNA released from other cells in suspension, likely during cell death. As with doublets, ambient RNA results in unexpected gene expression patterns. Most suggested analysis pipelines include ambient RNA correction before doublet detection however no formal investigation into how doublet detection is affected by ambient RNA correction has been conducted. Here, we evaluate the impact of ambient RNA correction on the performance of doublet detection using novel engineered and multiplexed datasets. We apply three of the most widely-used methods for removing ambient RNA ( decontX , soupX , CellBender ), in conjunction with up to six doublet detection methods ( scDblFinder , scds , bcds , cxds, scrublet , DoubletDetection ). We find that doublet detection tools perform best when done prior to ambient RNA correction, with the exception of CellBender . Performing ambient RNA correction first, particularly for decontX , leads to an increased proportion of doublet droplets retained in the dataset compared with calling doublets on uncorrected data. We show that both decontX and soupX partially mask doublet signal by removing proportionally more ambient RNA from doublets that remain undetected after ambient RNA correction. Our results show that the performance of doublet detection is significantly impacted by the use of ambient RNA correction tools. We suggest a new workflow that implements doublet detection prior to ambient RNA correction unless using CellBender .

Summary: Co‐designed research in paediatric HSCT is limited. We sought to determine research priorities which represent the shared priorities of patients, parents, carers and healthcare professionals (HCP) within Australia, New Zealand, the Netherlands and the United Kingdom. An international, multiphase priority‐setting methodology was implemented in partnership with the James Lind Alliance and delivered over an 18‐month period. Part 1: an international scoping survey asked respondents to submit their research uncertainties related to paediatric HSCT. Part 2: summarising and evidence‐checking the submitted uncertainties. Part 3: interim prioritisation survey. Part 4: consensus workshop. In the first international scoping survey, 667 topic ideas were suggested (45% by consumers, 55% by HCP), which were categorised into 80 summary questions. After systematic literature review, 35 summary questions were judged to be true uncertainties (i.e. not answered by existing evidence). These 35 uncertainties were included in a second interim prioritisation survey and completed by 224 participants. From those, a shortlist of 19 questions was drawn. After a multistakeholder workshop, consensus was reached on the top 10 priorities. The PSP identified important research gaps in the management of paediatric HSCT. Priority areas included: implementing personalised medicine approaches, improving immune recovery and adjunct interventions such as exercise, nutrition and microbiome‐directed strategies.

Objectives: To compare the measurement properties of paediatric patient-reported outcome measures (P-PROMs) used in clinical trials and practice for common conditions. Design: Data from the Australian Paediatric Multi-Instrument Comparison study were used, including children aged 5–18 years with autism spectrum disorder (ASD) (n=510), asthma (n=487), eating disorder(s) (n=216), epilepsy (n=298), recurrent abdominal pain (n=392), sleep problems (n=346), dental problems (n=490), type 1 diabetes (T1D) (n=67), enuresis (n=182) and no health conditions (reference group) (n=1259). The acceptability, response distribution (floor/ceiling effects), known-groups validity, test-retest reliability, responsiveness and convergent validity of the Paediatric Quality of Life Inventory (PedsQL), EuroQol Group’s Youth instruments with three and five response options (EQ-5D-Y-3L and EQ-5D-Y-5L), Child Health Utility 9D (CHU9D), Assessment of Quality of Life 6D (AQoL-6D), Health Utilities Index Mark 3 and Patient-Reported Outcomes Measurement Information System Paediatric Profile 25 (PROMIS-25) were assessed. Results: PedsQL, EQ-5D-Y-3L, EQ-5D-Y-5L, CHU9D and PROMIS-25 were easy and quick to complete, indicating acceptability. Only the EQ-5D-Y-3L and EQ-5D-Y-5L demonstrated ceiling effect issues. All measures were able to differentiate between children with no health conditions and children with each condition (p<0.001, effect sizes between 0.47 and 3.5), indicating known-groups validity. Test-retest reliability varied by condition; however, the PedsQL and EQ-5D-Y-5L were the most reliable. Conclusions: Certain P-PROMs may be preferred in different conditions, that is, ASD (PedsQL and EQ-5D-Y-5L), asthma (PedsQL), eating disorder(s) (PedsQL and CHU9D), epilepsy (PedsQL and CHU9D), recurrent abdominal pain (PedsQL, EQ-5D-Y-5L and CHU9D), sleep problems (PedsQL), dental problems (PedsQL), T1D (PedsQL and CHU9D) and enuresis (PedsQL). Notably, the PedsQL performed favourably in all conditions assessed. More evidence is required on the responsiveness of P-PROMs to changes in child health. Trial registration number: ACTRN12621000657820.

The lung alveoli are continually exposed to inhaled pathogens and environmental hazards and rely on coordinated communication between alveolar macrophages and type 2 alveolar epithelial cells (AT2s) to maintain homeostasis. Disruption of these interactions can impair immunity and repair, contributing to acute and chronic respiratory diseases. To better define these mechanisms and support therapeutic discovery, we established a human iPSC-derived air-liquid interface platform that captures key features of AT2-macrophage crosstalk. Using this system, we show that coculture enhances AT2-specific transcriptional programs including lipid synthesis, while macrophages actively phagocytose AT2-derived surfactant. iPSC-derived macrophages adopt an alveolar macrophage–like phenotype and respond to AT2-derived M-CSF. During respiratory infection, macrophages play a crucial role in modulating epithelial inflammatory responses, augmenting antiviral immunity, and limiting viral replication. We further identify a role for macrophages in epithelial repair, where VEGF-mediated signaling to macrophages increases epithelial permeability during viral infection. Together, these findings reveal dimensions of AT2-macrophage cooperation in homeostasis, infection, and repair, and demonstrate how this iPSC-derived platform can be used to dissect mechanisms that may initiate or drive the progression of respiratory diseases. Human stem-cell-based lung model was developed to investigate how immune cells and alveolar cells work together to fight infection, and coordinate repair lung damage.

Genome-wide association studies (GWAS) have discovered thousands of genetic variants linked to autoimmune disease, and yet the molecular pathways underlying autoimmunity have remained elusive. A key challenge is that >90% of identified GWAS risk loci are in non-coding genomic regions making it difficult to predict their relevance to disease. Here, we have curated fine-mapped non-coding risk variants from over 30 different autoimmune traits including common conditions such as systemic lupus erythematosus (SLE), Crohn’s disease, and multiple sclerosis, and reveal shared genetic signatures between diverse autoimmune diseases. We subsequently performed a high-throughput single-cell multi-omic CRISPR activation screen targeting 763 autoimmune risk loci in primary human B cells (a highly relevant cell type to autoimmune diseases) and discover 524 cis -regulatory target gene effects for 378 risk loci, with many risk loci regulating multiple gene targets. This Single Cell Analysis of Non-coding Distal Autoimmune Loci (SCANDAL) provides a powerful experimental resource linking non-coding risk loci to many disease-relevant genes, including lowly-expressed cytokines and transcription factors for which perturbation effects can be difficult to quantify with other CRISPR-based strategies. We reveal how increased transcriptional activity at one non-coding risk locus can drive transcription at other risk loci within the same regulatory landscape that may be relevant to understand genetic pleiotropy of autoimmune diseases. Finally, we quantified allele-specific effects on target gene expression with massive parallel reporter assays and prime editing to discover a gain-of-function variant associated with SLE that controls expression of the transcription factor REL /cREL which subsequently binds dozens of risk loci and target genes associated with different autoimmune diseases. Our study provides a valuable resource linking non-coding risk loci with their cis -regulatory target genes and advances our understanding of the shared genetic networks and mechanisms involved in autoimmunity.

Background: Pulmonary exacerbations pose a significant clinical burden on people with cystic fibrosis (pwCF). Whether management of exacerbations should change in the context of modulator therapy is unclear. We describe the characteristics, treatment and lung function outcomes of pulmonary exacerbations requiring intravenous antibiotic therapy (PERITs) in a contemporary Australian cohort of pwCF, in an era of rapidly broadening access to modulator therapy. Methods: PwCF receiving care at 11 Australian specialist centres were prospectively enrolled between 14 October 2020 and 9 October 2024. Spirometry data and treatments received during a PERIT were collected systematically. Results: A total of 982 pwCF were enrolled, with 593 PERITs recorded in 323 individuals. The median (interquartile range) age at PERIT start was 12 (7–17) years and the mean± sd baseline forced expiratory volume in 1 s (FEV ₁ ) % predicted across PERITs was 80±21%. Approximately 62% (n=366) of PERITs occurred in people receiving modulator therapy. Intravenous tobramycin (63%) and piperacillin-tazobactam (43%) were the most frequently used antibiotics. Among participants with spirometry at baseline and at Day 7 (n=296) or Day 60 (n=383), 41% (n=120) and 44% (n=169) were below their baseline at Day 7 and Day 60 after commencing treatment, respectively; 8% were >10% below their baseline FEV ₁ % predicted at both time-points. Recovery patterns were consistent regardless of baseline lung function, Pseudomonas aeruginosa colonisation or modulator use. Conclusion: The pattern and magnitude of lung function impairment during PERITs is similar among those receiving and not receiving modulator therapy. This underscores the continued need for evidence to help clinicians balance treatment burden against the risk of irreversible loss of lung function.

Aims: Asthma affects > 10% of children in Australia and New Zealand (NZ), with up to 5% of those having severe disease, presenting a management challenge. We aimed to survey tertiary paediatric respiratory services across Australia and NZ using a custom‐designed questionnaire, to conduct a cross‐sectional observational study of the numbers of children with problematic severe asthma (PSA) seen, the number treated with biologic therapy, outpatient clinic/multidisciplinary team (MDT) services available, investigations and tools routinely used and approaches utilised for transition to adult care. Methods: A custom‐designed online survey was distributed via email to Directors of public paediatric respiratory services across Australia and NZ ( n = 14). Reminders to prompt completion were emailed regularly over 3 months. Results: All sites provided survey responses. The estimated number of children with PSA across 12 sites was 561 (53 prescribed biologic treatment); two sites felt unable to provide accurate estimations. Most sites ( n = 8) did not have a MDT approach either as MDT clinics or meetings; patients were managed in either asthma ( n = 7, 50%) or general respiratory clinics. Most sites (85%) utilised questionnaires regarding asthma control for assessment, although some utilised additional questionnaires. Remaining tools and investigations varied widely across centres. Only four sites (29%) had established processes for transition to adult care. Conclusions: Given notable heterogeneity in service availability and PSA management across tertiary sites, children may experience variability in care dependent on their location. Most centres lack MDT models of care, which are considered the international best‐practise standard for management of children with PSA.

Introduction: Pulmonary complications due to infection contribute significantly to post‐haematopoietic stem cell transplant (HCT) morbidity and mortality. Standard microbiological investigations, when performed on bronchoalveolar lavage (BAL) fluid, can take days to weeks to confirm a diagnosis. We aimed to determine the diagnostic performance of the BioFire FilmArray pneumonia panel plus (FA‐PP), a multiplex polymerase chain reaction (PCR) panel that detects 18 bacterial and nine viral targets, when applied to BAL obtained by flexible bronchoscopy, in children undergoing HCT.Methods: We performed a single‐centre prospective observational study in children undergoing allogeneic HCT, who underwent BAL pre‐ and post‐HCT. To determine the diagnostic performance of the FA‐PP, we measured the positive and negative concordance, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), compared with standard microbiological investigations, which was considered the gold standard. The clinical impact of the FA‐PP was also qualitatively measured.Results: This study enrolled 16 children who had 31 BAL samples collected, both pre‐ and post‐HCT. In total, there were seven patients who underwent eight BALs while symptomatic, and 50% (4/8) of these results were concordant between FA‐PP and standard microbiological investigations. In the 13 patients who had 23 BALs collected while asymptomatic, 78% (18/23) of these results were concordant. In the cohort as a whole, sensitivity, specificity, PPV and NPV were 100%, 78%, 54% and 100%, respectively. In 50% (4/8) of patients who underwent a BAL while symptomatic, the FA‐PP resulted in a hypothetical or actual clinical change, compared to 22% (5/23) of patients who underwent an asymptomatic BAL.Conclusion: In conclusion, we report the first prospective evaluation of the diagnostic performance of the FA‐PP in BAL, in a high‐risk paediatric HCT cohort. We demonstrate that the FA‐PP is a potentially useful adjunct to traditional standard microbiological investigations that can provide clinically impactful diagnostic information.