Melbourne Bioinformatics

Drosophila immunity has been the focus of intense study and has impacted other research fields including innate immunity and agriculturally or epidemiologically relevant investigations of insect pests and vectors. Unsurprisingly for such a large body of work, some published results were later found to be irreproducible. Although some results have been contradicted in the literature, many have no published follow-up, either due to a lack of research or low motivation to publish negative or contradictory results. We have addressed this by performing a reproducibility project that analyses the verifiability of claims from articles published on Drosophila immunity before 2011. To assess reproducibility, we extracted claims from 400 articles on the Drosophila immune response to bacteria and fungi and performed preliminary verification by comparing these claims to other published literature in the field. Using alternative approaches, we also experimentally tested some ‘unchallenged’ claims, which had no published follow-up. The intent of this analysis was to centralize evidence on insights and findings to improve clarity for scientists that may base research programs on these data. All our data are published on a publicly available website associated with this article (https://ReproSci.epfl.ch/) that encourages community participation. This article provides a short summary of claims that were found to have contradictory evidence, which may help the community to assess past findings on Drosophila immunity and improve clarity going forward.

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a major cause of glomerular disease that can rapidly progress to end-stage kidney disease. Lack of safe and effective treatment represents a significant unmet need for patients with FSGS. Development of treatments for FSGS has been hampered by complexities including the rare nature of the disease, heterogeneity of disease pathogenesis, variable disease course and ongoing debate regarding appropriate FSGS trial endpoints. DMX-200 (repagermanium) is a C-C motif chemokine receptor 2 (CCR2) pathway inhibitor. Activation of the angiotensin II type 1 receptor (AT1R) and CCR2 have been independently implicated in the pathogenesis and progression of FSGS. Both these receptors form a functional complex, so inhibition of both receptors may provide an efficient approach to FSGS treatment. In vivo data shows co-administration of DMX-200 and the angiotensin receptor blocker (ARB) irbesartan demonstrated synergistic reductions in proteinuria, macrophage infiltration, and podocyte loss compared with vehicle or either antagonist alone. A subsequent Phase 2a placebo controlled, cross-over study (NCT03649152) in n = 8 patients with biopsy-proven primary FSGS showed that the combination of DMX-200 and irbesartan was well tolerated with most patients experiencing a reduction in proteinuria. Based on these data, the global Phase 3 study (ACTION3) was initiated to confirm the proteinuria lowering effects of DMX-200 and assess effects on eGFR decline. ACTION3 is a randomised, placebo-controlled, double-blind study of FSGS patients and, on completion, will provide a resource for the wider nephrology community and advocacy groups on the natural history of FSGS patients and the effects of a CCR2-inhibitor on the background of an ARB. Here we present the baseline data from the first 144 patients (50% to target) enrolled in ACTION3 as of 1st January 2025. Method The ACTION3 (NCT05183646) study is a pivotal 104 week Phase 3, multi-centre study of the efficacy and safety of DMX-200 administered 120 mg twice daily compared with placebo in patients with FSGS concomitantly receiving maximally tolerated ARB. The primary objective is to evaluate the efficacy of DMX-200 using percent change in 24-hour urine PCR and eGFR slope. Key eligibility criteria are a diagnosis of FSGS (primary, genetic or undetermined cause), persistent proteinuria >1.5 g/day and eGFR >25 ml/min/1.73 m2. The study plans to enrol approximately 286 adult patients. The initial 104-week randomised double-blind period will be followed by a 2-year open label extension study. The trial is reviewed on a regular basis by the Independent Data Monitoring Committee (IDMC). Results The study is actively recruiting at over 200 sites in 21 countries (Table 1) and passed 50% recruitment (n = 144 patients) in late December 2024. Recruitment of paediatric patients (12–17 years old) will occur at approximately 19 sites in Argentina, Mexico, United Kingdom and the United States with the first paediatric patient recruited in January 2025. Demographic data of the first 144 randomised patients is listed in Table 2. Patients are predominantly male (64.6%), with an average age of 45.4 yrs. and mean eGFR 58.8 ± 27.86 (ml/min/1.73 m2). The ACTION3 IDMC has now completed 5 reviews of the study data including a futility analysis that found the study non-futile (March 2024). As of January 2025, the mean duration of treatment with DMX-200 or placebo is now in excess of 52 weeks. In September 2024, the first patient entered the open label extension, with 14 patients enrolled as of January 2025. No clinically significant safety concerns have been observed to date. Conclusion DMX-200 (repagermanium) has shown encouraging signs of safety and efficacy in combination with an ARB for the treatment of FSGS. The contributing countries and baseline characteristics of the patients enrolled in ACTION3 are similar to those reported in other recent FSGS trials. No clinically significant safety concerns have been observed in patients enrolled to date in the ACTION3 Phase 3 study.

The bacterial genus Endozoicomonas is a predominant member of the coral microbiome, widely recognised for its ubiquity and ability to form high-density aggregates within coral tissues. Hence, investigating its metabolic interplay with coral hosts offers critical insights into its ecological roles and contributions to coral health and resilience. Using long- and short-read whole-genome sequencing of 11 Endozoicomonas strains from Acropora loripes, genome sizes were found to range between 5.8 and 7.1 Mbp. Phylogenomic analysis identified two distinct clades within the family Endozoicomonadaceae. Metabolic reconstruction uncovered clade-specific pathways, including the degradation of holobiont-derived carbon and lipids (e.g., galactose, starch, triacylglycerol, D-glucuronate), the latter of which suggests involvement of Endozoicomonas in host 'sex-type' steroid hormone metabolism. A clade-specific type 6 Secretion System (T6SS) and predicted effector molecules were identified, potentially facilitating coral-bacterium symbiosis. Additionally, genomic analyses revealed diverse phosphorus acquisition strategies, implicating Endozoicomonas in holobiont phosphorus cycling and stress responses. This study reveals clade-specific genomic signatures of Endozoicomonas supporting its mutualistic lifestyle within corals. Findings suggests possible roles in nutrient cycling, reproductive health, and stress resilience, offering novel insights into coral holobiont functioning.

The dynamic nature of human endometrial tissue presents unique challenges in analysis. Despite extensive research into endometrial disorders such as endometriosis and infertility, recent systematic reviews have highlighted concerning issues with the reproducibility of omics studies attempting to identify biomarkers. This review examines factors contributing to poor reproducibility in endometrial omics research. Hormonal fluctuations in the menstrual cycle lead to widespread molecular changes in the endometrium, most notably in gene expression profiles. In this review, we examine the variability in omics data due to the menstrual cycle and highlight the importance of accurate menstrual cycle dating for effective statistical modelling. The current standards of endometrial dating lack precision and we make the case for using molecular-based modelling methods to estimate menstrual cycle time for endometrium tissue samples. Additionally, we discuss statistical considerations such as confounding and interaction effects, as well as the importance of recording the detailed and accurate clinical information of patients. By addressing these methodological challenges, we aim to establish more robust and reproducible research practises, increasing the reliability of endometrial omics research and biomarker discovery.

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease.

Abstract In recent years, the potential to locate endangered animals using scent trained detection dogs (conservation detection dogs) has gained attention. Among vertebrates, conservation detection dogs have demonstrated a remarkable capacity to detect the scent of endangered mammals, reptiles, and birds, but their use in detecting amphibians is only beginning to be explored. A lack of work in this area is surprising given that amphibians are declining faster than any other vertebrate taxa. Moreover, amphibians are generally small, highly cryptic and breed in complex habitats, making them difficult to locate for the purpose of monitoring or establishing conservation breeding programs. This study aimed to provide a preliminary investigation into whether conservation detection dogs can be imprinted on the scent of the critically endangered Baw Baw frog (Philoria frosti) under captive conditions, and then effectively trained to locate wild frogs in their complex natural habitat. Two conservation detection dogs were trained to identify and locate P. frosti scent under controlled conditions before assessing their ability to locate wild P. frosti. Both conservation detection dogs were effective at locating P. frosti scent under controlled conditions, and also demonstrated an ability to detect live frogs under natural conditions. From an applied perspective, our findings provide new evidence that conservation detection dogs are capable of learning to detect the scent of small, cryptic anuran species. They also indicate that detection dogs are capable of locating frogs in highly complex forest habitat, confirming their untapped potential to aid in the management of imperiled species that have previously proven difficult to detect, monitor, and protect. We discuss the limitations of our approach and provide recommendations to help direct future amphibian conservation detection dog research.

Non-obstructive azoospermia (NOA) diagnosis poses challenges for couples seeking parenthood. Microdissection testicular sperm extraction (MD-TESE) excels in retrieving testicular sperm cells for NOA cases. However, limited live birth data in Australian NOA patients hinders accurate counselling. This study aimed to determine the likelihood of infertile couples with a male partner diagnosed with NOA conceiving biological children using MD-TESE / intracytoplasmic sperm injection (ICSI). A retrospective cohort study included 108 NOA men treated at a public fertility unit and a private fertility centre (May 2009-May 2022). live birth rate (LBR); secondary outcomes: sperm retrieval rate, pregnancy rate, and neonatal outcomes. Among 108 patients undergoing MD-TESE, the positive sperm retrieval rate (PSRR) was 64.8% (70/108). Histology best predicted sperm retrieval success, with hypo-spermatogenesis yielding a 94.1% PSRR. Age, testicular volume, and hormonal parameters had no significant impact. Mean male age: 35.4 years; mean partner age: 32.7 years. Fertilisation rate: 50.7%. LBR per initiated cycle: 58.7% (37/63); per embryo transfer: 63.8% (37/58); per initially diagnosed NOA man: 34.3% (37/108). Cumulative LBR: 74.1% (43/58); twin rate: 10.8% (4/37). No neonatal deaths or defects were observed among 47 live offspring. This study provides valuable data for counselling NOA couples on the probability of conceiving biological offspring. MD-TESE and ICSI yielded favourable PSRR (64.8%) and LBR (63.8%). However, couples should be aware that once NOA is confirmed, the chance of taking home a baby is 34%.

The microbiome has long been suspected of a role in colorectal cancer (CRC) tumorigenesis. The mutational signature SBS88 mechanistically links CRC development with the strain of Escherichia coli harboring the pks island that produces the genotoxin colibactin, but the genomic, pathological and survival characteristics associated with SBS88-positive tumors are unknown. SBS88-positive CRCs were identified from targeted sequencing data from 5,292 CRCs from 17 studies and tested for their association with clinico-pathological features, oncogenic pathways, genomic characteristics and survival. In total, 7.5% (398/5,292) of the CRCs were SBS88-positive, of which 98.7% (392/398) were microsatellite stable/microsatellite instability low (MSS/MSI-L), compared with 80% (3916/4894) of SBS88 negative tumors (p=1.5x10-28). Analysis of MSS/MSI-L CRCs demonstrated that SBS88 positive CRCs were associated with the distal colon (OR=1.84, 95% CI=1.40-2.42, p=1x10-5) and rectum (OR=1.90, 95% CI=1.44-2.51, p=6x10-6) tumor sites compared with the proximal colon. The top seven recurrent somatic mutations associated with SBS88-positive CRCs demonstrated mutational contexts associated with colibactin-induced DNA damage, the strongest of which was the APC:c.835-8A>G mutation (OR=65.5, 95%CI=39.0-110.0, p=3x10-80). Large copy number alterations (CNAs) including CNA loss on 14q and gains on 13q, 16q and 20p were significantly enriched in SBS88-positive CRCs. SBS88-positive CRCs were associated with better CRC-specific survival (p=0.007; hazard ratio of 0.69, 95% CI=0.52-0.90) when stratified by age, sex, study, and by stage. SBS88-positivity, a biomarker of colibactin-induced DNA damage, can identify a novel subtype of CRC characterized by recurrent somatic mutations, copy number alterations and better survival. These findings provide new insights for treatment and prevention strategies for this subtype of CRC.

In this study, the composition of amino acids, nutritional characteristics, degree of hydrolysis (DH), antioxidant properties, and antibacterial activity of proteins and hydrolysates of bellflower (Campanula latifolia), Persian willow (Salix aegyptiaca), and bitter orange (Citrus aurantium L.) were investigated under the influence of different proteases (Alcalase: Al, trypsin: Tr, pancreatin: Pa, and pepsin: Pe). Evaluation of the structural features of the proteins showed amide regions (amide A, B, I-III) and secondary structures. Hydrophobic amino acids (∼38%), antioxidants (∼21%), and essential types (∼46%) form a significant part of the structure of flower pollen. The digestibility and nutritional quality (PER) of the hydrolyzed samples (CP: 1.67; CA: 1.89, and PW: 1.93) were more than the original protein. Among proteins and peptides, the highest degree of hydrolysis (34.6%: Al-PWH), inhibition of free radicals DPPH (84.2%: Al-CPH), ABTS (95.2%: Pa-CPH), OH (86.7%: Tr-CAH), NO (57.8%: Al-CPH), reducing power (1.31: Pa-CPH), total antioxidant activity (1.46: Pa-CPH), and chelation of iron ions (80%: Al-CPH and Al-CAH) and copper (50.3%: Pa-CAH) were affected by protein type, enzyme type, and amino acid composition. Also, the highest inhibition of the growth of Escherichia coli (25 mm) and Bacillus cereus (24 mm) were related to CP and PW hydrolysates, respectively. The results of this research showed that hydrolyzed flower pollens can be used as a rich source of essential amino acids as well as natural antioxidants and antibacterial in food and dietary products. PRACTICAL APPLICATION: Enzymatic hydrolysis of Campanula latifolia, Persian willow, and Citrus aurantium pollen proteins was performed. The hydrolyzed ones had high nutritional quality and digestibility (essential amino acids and PER index). Antioxidant properties and chelation of metal ions of peptides were affected by the type of protein and enzyme. The hydrolysates showed inhibitory activity against the growth of Esherichia coli and Bacillus cereus.