Biology subjects

Mason, C.

Publications and source records attributed to Mason, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Simulated microgravity induces aging phenotypes in human organoids

Simulated microgravity provides a controlled perturbation for testing how loss of mechanical load affects human tissues. After 24 hours in rotating-wall vessels, neural, cardiac and tonsil organoids developed tissue-specific transcriptional responses that overlapped published spaceflight datasets, accompanied by higher transcriptomic-age estimates in neural and cardiac models, altered cardiac-organoid contraction dynamics and modified tonsil-organoid antiviral gene-expression signatures. These findings establish a multi-organoid framework for studying acute responses to gravitational unloading.

molecular biology↗

bioRxiv2026

CellCage™ Technology Enables High-throughput Isolation of Defined Cell Combinations to Resolve the Determinants of CAR-T mediated Cytotoxicity

Understanding the cellular determinants of CAR-T cell-mediated cytotoxicity at the single-cell level remains a critical challenge in developing effective cellular immunotherapies. Conventional bulk co-culture assays provide only population-level measurements of cytotoxic activity and cannot resolve the functional heterogeneity of individual effector-target interactions. Here, we demonstrate the Cellanome platform, which enables the formation of CellCage Enclosures (CCEs) through spatially controlled photopolymerization, as a tool for high-resolution, high-throughput characterization of CAR-T cell-mediated cytotoxicity. By isolating defined effector-target combinations within individual CCEs and coupling encapsulation with longitudinal time-lapse imaging and automated image analysis, we resolve cytotoxic activity across thousands of individually tracked CCEs. Using anti-CD19 CAR-T effectors and NALM6-GFP targets, we show that individual CD8+ CAR-T effectors exhibit substantial functional heterogeneity, with serial killing capacity, effector-target contact dynamics, and intrinsic motility each independently correlating with cytotoxic potency. Cytotoxic efficacy scaled positively with effector number, and CD4+ T helper cells augmented CD8+-mediated killing in a dose-dependent manner, providing direct evidence for cooperative T cell behavior during tumor cell clearance. Extending the platform to a solid tumor model using AIC100 anti-ICAM-1 CAR-T cells and HeLa-GFP targets, we show that individual AIC100 effectors are insufficient to mediate effective killing, while multi-effector CCEs exhibit robust cytotoxicity scaling with effector abundance and CD4+:CD8+ composition. These data establish the Cellanome platform as a broadly applicable system for dissecting the determinants of CAR-T cell cytotoxicity at single-cell resolution, with direct translational relevance to the optimization of cellular immunotherapy.

immunology↗

bioRxiv2026

Sampling design and inference of the caecal-skin Campylobacter relationship in broilers

The relationship between Campylobacter levels in broiler caeca and on carcass skin is central to quantitative microbial risk assessment along the poultry production chain, underpinning modelling of intervention impacts, including EFSA assessments of the public health impact of control measures. However, this relationship is typically inferred from monitoring data generated under sampling designs that do not preserve pairing between specimens and may involve pooling. In this study, we used a simulation framework to evaluate whether commonly used sampling strategies allow reliable recovery of the caecal-skin relationship. A simulated broiler population was generated, assigning caecal and skin loads to individual birds based on a specified linear relationship. Sampling was conducted under paired and unpaired designs, with and without pooling, reflecting approaches used in surveillance programmes and in policy-oriented models. Regression models were fitted to sampled data across 1,000 simulations for a range of assumed slopes. Under paired sampling, estimated slopes closely matched the true relationship across most scenarios. In contrast, unpaired sampling consistently failed to recover the association, with estimated slopes centred around zero regardless of the true slope. These findings were robust to variation in within-flock prevalence, residual error, and intercept. The results show that sampling design fundamentally affects identifiability of relationships between stages of the production chain. This has implications for interpretation of parameters derived from monitoring data and used in quantitative Campylobacter risk assessments informing policy. Parameters derived from unpaired and pooled monitoring data should therefore be interpreted with caution when used to support risk assessment and decision-making. Campylobacter; broiler chickens; sampling strategy; unpaired sampling; carcass contamination; quantitative microbial risk assessment; simulation.

microbiology↗

bioRxiv2026

Simulated spaceflight disrupts the immune-gut-brain axis and drives sex-dependent neuroinflammation, axonal injury, and behavioral deficits.

Simulated spaceflight perturbs multiple organ systems, yet the integrated impact of spaceflight-relevant stressors on the immune-gut-brain axis remains poorly defined. We used a ground-based model combining hindlimb unloading (HU) with low-dose ionizing radiation (IR; 50 or 100cGy) to quantify neuropathology, peripheral immune phenotypes, intestinal barrier integrity, and behavioral performance in male and female C57BL/6 mice. HU and/or IR induced region-selective neurodegenerative changes consistent with axonal injury across the cortex and major white-matter tracts. In the somatosensory cortex, MAP-2+ neurons were reduced and SMI-312-labeled axonal injury increased, lowering the intact-to-dystrophic axonal area ratio. Long-range fiber pathways (corpus callosum, cingulate gyrus, external capsule) showed robust axonal damage accompanied by gliosis, with elevated Iba-1+ microglia and GFAP+ astrocytes most prominent after HU+IR (100cGy). Peripheral immunophenotyping revealed a sustained, sex-dependent innate inflammatory bias, with expanded CD11b+ myeloid cells and increased TNF-+ myeloid activation after IR and IR+HU, alongside maladaptive T-cell polarization despite largely unchanged total CD8+ and CD4+ frequencies. In parallel, the gut exhibited architectural remodeling and barrier failure, including altered mucin profiles, reduced ZO-1 tight-junction labeling, and increased CD45+ leukocyte infiltration across the jejunum, ileum, and colon. Behavioral assays demonstrated sex-dependent deficits spanning affective, motor, and cognitive domains, including increased anxiety- and depressive-like behaviors, impaired rotarod performance, reduced recognition memory, and less efficient spatial strategies. Overall, these findings identify a sex-dependent immune-gut-brain vulnerability in which combined HU and low-dose IR drive gut barrier breakdown and immune imbalance that coincide with neuroinflammatory axonopathy and measurable neurobehavioral dysfunction.

neuroscience↗

bioRxiv2026

Leveraging quadplexed digital PCR to characterize gene therapy vectors

Currently there is a lack of high-throughput, low material-input methods to screen early-stage product quality of viral and non-viral gene therapy products. Here we propose using multiplex droplet digital PCR (dPCR) to screen and characterize vector sequences. We describe the adaptation of a Poisson-multinomial model to quantitate integrity of any combination of 4 targets in multiplexed ddPCR. We show the success and limitations of model employment and provide some suggested best practices.

molecular biology↗

bioRxiv2026

Disrupted glial-mediated synaptic refinement in Fragile X syndrome

Fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism, results from the loss of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP is a translational regulator and is highly expressed in glial cells, where its role in neural circuit development remains poorly defined. Here, it was observed that Fmr1 knockout mice exhibit reduced synapse size and accelerated eye-specific segregation. To examine which cell-types participate in this process, a multi-omic framework was applied to FXS model mice at postnatal day 7, a critical window for synaptic remodeling in the retinogeniculate pathway, an established model system utilized to study synaptic pruning. Single-cell transcriptomics revealed coordinated alterations in microglia, astrocytes, and neurons in genes linked to synaptic pruning. Computational modeling further demonstrated enhanced astrocyte-to-microglia signaling, particularly through Ephrin A (EphA)- and semaphorin-mediated pathways, while lipidomic profiling revealed reductions in EphA-associated lipid species required for lipid raft stability and receptor localization. Consistent with these observations, a glial engulfment assay indicated that FXS microglia and astroglia over-engulf synaptic material in the lateral geniculate nucleus, supporting the transcriptomic profile. Together, these findings identify impaired glial-driven synaptic refinement as an early mechanistic feature of FXS pathogenesis, highlighting the genes involved in this process as potential therapeutic targets during circuit development.

neuroscience↗

bioRxiv2026

Integrating coastal microbiome observations for human, oyster and environmental protection

The Reseau dObservatoires de Microbiologie Environnementale integree (ROME) was a pilot study conducted in France from September 2020 to August 2023 aiming to establish a network of eDNA-based observatories across four estuarine ecosystems associated with oyster farming: the Bay of Veys (Normandy), the Bay of Brest (Brittany), Marennes-Oleron (Nouvelle-Aquitaine), and the Thau Lagoon (Occitania). Within a One Health framework, the study assessed the influence of river inputs on estuarine microbiome structuring and the emergence of microbiological hazards affecting human, aquaculture, and ecosystem health. Over 2,000 samples were collected during the study, including biweekly surface water and monthly adult oyster samples. Environmental nucleic acids were analysed using metabarcoding (bacterial and protist communities) and metagenomics (human RNA viruses). The coastal microbiome, including pathogenic and harmful taxa relevant to humans and aquatic invertebrates, was characterized. River influence on microbial community composition was examined through spatial comparisons of stations exposed to varying levels of freshwater runoff, while oysters acted as bio-integrators of local microbial diversity. Results revealed coherent coastal-to-offshore microbiome structuring across all ecosystems, with local variations linked to riverine inputs. eDNA metabarcoding allowed to detect a wide range of prokaryotic and eukaryotic pathogens, as well as harmful algal bloom (HAB) genera, several not captured by conventional monitoring. These findings demonstrate the potential of the ROME eDNA observatory network for high-resolution, integrative surveillance of microbial biodiversity and early detection of biological risks in estuarine environments.

genomics↗

bioRxiv2026

Whole-genome benchmarking reveals context-specific error rates in the Ultima UG100 and Illumina NovaSeqX Platforms.

Whole genome sequencing (WGS) is becoming more common in research and clinical applications, but there is a paucity of comparative data between high-throughput WGS platforms. We benchmarked the Ultima Genomics UG100 system against Illumina NovaSeqX using the Genome in a Bottle (GIAB) WGS reference set sample HG002. Across NIST v4.2.1 benchmark regions, UG100 exhibited higher total variant-calling errors (27x), primarily driven by indel false negatives. Restricting to Ultima Genomics high confidence regions (UG HCR v3.1, 90.3% of GRCh38) reduced the error burden by 89.6%, indicating most errors lie outside these regions. Strong degradation of variant calling performances occurred in homopolymer tracts > 10 bp, and base calling error rates increased sharply after position 200 bp within each read, whereas Illuminas error rates were higher earlier in the read. Coverage dropouts in UG100 data were pronounced in GC-rich regions. Of note, 2.24% of ClinVar Pathogenic and Likely Pathogenic variants and 22.6% of a common catalogue of polymorphic STRs were found to be excluded from UG HCR v3.1, indicating potential impact to clinical applications. These results highlight context-specific genotyping errors of the UG100 and NovaSeqX platforms, and underscore the importance of whole genome benchmarking for adjudicating accuracy of NGS platforms beyond the current HG002 reference set.

genomics↗

bioRxiv2026

Cryptic Insect Microbiome Compositions Unveiled with Full-Length 16S Sequencing

Insects have interconnected relationships with gut microorganisms which can have impacts on behavior and survival. Understanding foundational ecological principles of the insect-associated microorganisms is important to understand how insects utilize microbes to cope with stress and different environments. Common microbiome surveys of insect microbiomes utilize 16S rRNA sequencing of metagenomic DNA. For amplicon-based surveys of insect microbiome, fragment length and 16S rRNA subunit choice may have unintended biases, and some primer combinations may under-represent genus or species richness. Contemporary solutions target sequencing of the entire 16S rRNA fragment. Here, we illustrate the benefit of full-length 16S rRNA sequencing in improving sample resolution compared to V4 which provides new insight into the gut microbiome community composition of an invasive insect. We evaluated the gut microbiome of mass-reared medfly males (Mediterranean fruit fly, Ceratitis capitata) that were collected across a nine-month sampling period. Full-length 16S rRNA PCR products of samples were prepared into a Kinnex 16S rRNA library, sequenced on a PacBio Revio system, and the resulting HiFi amplicon data was processed into amplicon sequence variants (ASVs). Our findings reveal substantial differences in bacterial compositions across different medfly cohorts when sampling the full-length amplicons which were not detectible when only considering the V4 regions. Strain-level gut microbiome variation were supported with genomes assembled from shotgun metagenomic sequencing. Our findings support that long-read sequencing of full-length 16S rRNA amplicon uncover ecologically important interactions between host and gut microbiomes and serve as a bridge between short-fragment sequencing and shotgun metagenomics.

microbiology↗

bioRxiv2026

Applying PCR cycle autonormalization to improve PacBio full-length 16S rRNA sequencing

The bacterial 16S rRNA gene is widely used to characterize host-associated and environmental microbiomes, most commonly through sequencing short hypervariable regions. Recent improvements in PacBio sequencing chemistry and concatenation approaches can now enable high-throughput, full-length 16S rRNA gene sequencing with high accuracy and depth. However, PCR-induced errors remain a major limitation, particularly for full-length amplicons where error accumulation may be elevated due to a five-fold increase in sequence lengths. These challenges are exacerbated when samples vary widely in microbial biomass, making it difficult to select a single optimal number of PCR cycles. Here, we evaluated the effects of PCR cycle autonormalization on PacBio Kinnex full-length 16S rRNA gene sequencing across seven agriculturally relevant specimen types. We compared conventional PCR protocols (20, 24, and 30 cycles) with an autonormalization approach in which individual reactions were terminated during exponential amplification based on real-time fluorescence thresholds. Autonormalized reactions consistently retained a higher proportion of sequences following denoising and chimera removal, exhibited fewer substitution-based errors, and produced more even read distributions across samples. Meanwhile, overamplified reactions (30 cycles) showed elevated error rates and higher sequence removal, particularly in samples with greatest microbial biodiversity. Importantly, PCR protocol had minimal effects on overall community composition relative to specimen types. These results demonstrate that PCR cycle autonormalization improves data yield for full-length 16S rRNA sequencing while maintaining robust inferences, while facilitating streamlined workflows for heterogeneous samples. ImportanceAmplicon-based sequencing of the 16S rRNA gene is foundational tool in microbiome research, yet PCR amplification remains a major source of technical error. This challenge is magnified for full-length 16S rRNA sequencing and for workflows that process specimen types with widely varying microbial biomass. Selecting a single PCR cycle number can result in under-amplification of samples with low 16S rRNA copies or over-amplification and error accumulation in high-titer samples. Here, we show that PCR cycle autonormalization, implemented using iconPCR, substantially improves PacBio full-length 16S rRNA sequencing by reducing PCR-induced substitution errors and chimeras while maintaining consistent biological inference. Autonormalization also enables blind pooling of amplicons without post-PCR quantification or equimolar normalization, reducing hands-on time and sample loss. These benefits make cycle autonormalization particularly valuable for high-throughput and production-scale sequencing applications handling diverse specimen types.

microbiology↗

bioRxiv2026

Use of Metagenomic Reference Materials to Compare Lysis and Extraction Methods Against a Known Input

Inconsistent cellular lysis is a major source of inaccuracy in microbial analysis; there is a need to develop objective and standardized evaluation of lysis efficiency across diverse methods. Using a mock microbial community as a ground truth, we built a bioinformatic and experimental toolkit for this purpose, comparing the performance of chemical, thermal, enzymatic, and various physical lysis protocols. We evaluated over 150 lysis conditions and observed the highest performance from mechanically- and materially-optimized bead-based approaches. To extend these findings to a practical setting, we demonstrated that the Firmicutes to Bacteroides ratio, a frequently used compositional metric in gut microbiome analysis is biased heavily by insufficient lysis, indicating the importance of benchmarking ongoing methodological development.

microbiology↗

bioRxiv2025

Protamine lacunae preserve the paternal chromatin landscape in sperm

The transmission of the paternal genome requires extensive chromatin reorganization, in which nucleosomes are largely replaced by protamines that drive extreme condensation of the genome in the sperm head. Using Fiber-seq, we resolve patterns of paternal chromatin repackaging in sperm at single-molecule resolution, revealing the interplay between protamination and nucleosome retention. We find that nucleosome retention is probabilistic, with no locus universally occupied. Although promoters of spermatogenic genes preferentially harbor retained nucleosomes, the predominant carrier of paternal epigenetic information is protamine lacunae, accessible discontinuities in the protamine coat that preferentially mark critical regulatory elements. By contrast, centromere kinetochore binding regions robustly retain CENP-A mono-nucleosomes, providing a mechanism for the focal transmission of paternal centromeres. Finally, we find that paternal chromatin repackaging is altered in low-motility sperm. Together, these findings reveal distinct modes of paternal chromatin epigenetic inheritance with broad implications for development and infertility.

genomics↗

bioRxiv2025

Single-cell spatial mapping reveals dynamic bone marrow microarchitectural alterations and enhances clinical diagnostics in MDS

Myelodysplastic neoplasms (MDS) disrupt bone marrow hematopoiesis, yet clinical assessment relies largely on blast enumeration and qualitative morphology, which incompletely capture marrow architecture and disease state. We applied whole-slide multiplex immunofluorescence imaging with single-cell phenotyping to map bone marrow microarchitecture in MDS. Diagnostic biopsies (n=36), longitudinal treatment samples (n=29), precursor states (n=13), and normal controls (n=21) were analyzed, comprising >5 million spatially resolved cells. MDS marrow exhibited coordinated, genotype-imprinted architectural remodeling, including altered progenitor composition and spatial patterning, disrupted erythroid island organization, and displacement of hematopoietic stem and progenitor cells from perivascular niches. Interrogation of 82 cellular and spatial features yielded a composite Microarchitectural Perturbation Score (MDS-MAPS), derived from diagnostic samples and fixed prior to longitudinal analyses. In leave-one-patient-out cross-validation, MDS-MAPS discriminated remission from active disease more accurately than blast percentage (AUC 0.883 vs 0.660) and distinguished low-blast MDS from clonal cytopenia of undetermined significance (CCUS) (AUC 0.815). Mixed-effects modeling showed MAPS decreased in remission statistically independent of blast burden, with architectural normalization during remission and re-emergence at relapse. These findings define quantitative bone marrow architecture as a dynamic tissue-state biomarker that complements molecular and blast-based assessment in MDS.

pathology↗

bioRxiv2025

CCAAT-enhancer binding protein delta functions as a tumor suppressor gene in acute myeloid leukemia

There is a continued need for identification of novel disease drivers of acute myeloid leukemia as many patients experience relapse and have poor clinical outcomes. Analyses from our study and publicly available datasets predicted CEBPD as a novel tumor suppressor gene in acute myeloid leukemia. Consistent with the analyses, CEBPD knockdown experiments showed activation of MAPK signaling with concomitant increase in cell growth rate, while upregulation experiments suggested induction of myeloid differentiation marker CD14 expression in AML cell lines OCI-AML2 and OCI-AML5. Consistent with a previous report, our genomics analyses and azacytidine treatment experiments suggested a role for DNA methylation in downregulation of CEBPD expression during AML pathogenesis. Altogether, our results provide experimental evidence for a tumor suppressor function of CEBPD in AML.

cancer biology↗

bioRxiv2025

An loss of independence: genomic insights into a pest fruit fly-bacterial mutualism

Obligate microbial symbioses are often characterized by streamlined biosynthetic pathways and reduced genomes. The evolutionary process of this reduction first involves an increase in the abundance of non-functional coding genes (pseudogenes) followed by their removal. The olive fruit fly (Bactrocera oleae) harbors an extracellular symbiotic gut bacterium Candidatus Erwinia dacicola, which is crucial to its usage of fruit from the olive genus Olea as a larval food source. In this study, we combined genomics and transcriptomics of Ca. E. dacicola to investigate pathways that facilitate this mutualism. Of 4,675 genes in the Ca. E. dacicola genome, 1,783 were classified as pseudogenes. Some biochemical pathways such as amino acid pathways, biofilm regulator BssS, and 6-phospho-{beta}-glucosidase which are implicated in hydrolyzing oleuropein were complete. However, pathways connected to baseline homeostasis, which would impact cellular functions needed for a bacterium to be free-living, were heavily pseudogenized. Gene selection analyses in Ca. E. dacicola, when compared to related organisms, indicated positive selection on genes related to amino acid metabolism, carbon utilization, transport, and energy production. Our results indicate that Ca. E. dacicola is likely producing amino acids and metabolizing plant phytochemicals. These results reveal that the Ca. E. dacicola genome is undergoing incipient genome erosion in support of an unculturable obligate mutualism. ImportanceMany beneficial bacteria that live inside insects have highly reduced genomes, but little is known about the transitional stages that occur as free-living microbes evolve into obligate symbionts. We show that the olive fruit fly symbiont, Candidatus Erwinia dacicola, retains hallmarks of its plant-associated ancestry while undergoing extensive genome degradation, including the accumulation of mobile DNA elements and inactive genes. At the same time, genes involved in nutrient production, environmental persistence, and host interactions remain functional and are evolving under selection, providing a rare snapshot of how bacterial genomes are reshaped during the evolution of an obligate mutualism. These findings have implications for how obligate gut symbioses are formed and maintained in insect herbivores.

genomics↗

bioRxiv2025

Genome report: chromosome-scale genome assembly of the Olive fly Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae), is a specialist of fruits of the genus Olea and is a major pest of commercial olives due to their adverse impacts to olive production. In support of genomic and physiological research of the olive fly, we sequenced, assembled, and annotated two independent genomes, one from a wild-collected male and one from a wild-collected female. The resulting genomes are highly contiguous, collinear, and complete, attesting to the accuracy and quality of both assemblies. In addition to the autosomes captured as single contigs, the X and Y chromosomes were also captured as evidenced by the X chromosome showing diploid coverage in the female assembly compared to haploid coverage in the male assembly and the Y chromosome being entirely absent from the female assembly. These assemblies represent the first full chromosome-level assembly for Olive fly. In addition, a complete genome assembly of a known obligate symbiont to the olive fly, Candidatus Erwinia dacicola, was fully captured. The Ca. E. dacicola we report here is the most contiguous to date, represented with a gapless chromosome and two separate gapless plasmids. These genome assemblies, along with bacterial symbiont assembly, provide foundational resources for future genetic and genomic research in support of its management as an agricultural pest.

genomics↗

bioRxiv2025

Proteomic characterisation of Japanese quails unique seminal foam

Male Japanese quail (Coturnix japonica) produce a unique proctodeal foam upon ejaculation which is thought to enhance fertilisation success. However, the precise function and proteomic characterisation of this foam remain largely unexplored. Using high throughput LC-MS/MS proteomics analysis, we characterised the proteome of the quail seminal foam for the first time. Our analysis confidently identified 224 proteins in the foam using 32 pooled samples from 96 males. 96.4% of proteins had orthologs in the closely related (diverged 35 million years ago) chicken (Gallus gallus) which generated 40 Gene Ontology terms. Further interrogation revealed proteins involved in sperm motility, maturation and DNA protection, suggesting the foam plays a role in fertilisation success. Proteins linked to immune defence and inflammation modulation were identified, indicating the foam may protect sperm from antigens and reduce female immunogenic responses. This work advances our understanding of the function and evolution of Japanese quail unique seminal foam and directs future experiments to investigate the role of key seminal foam proteins in post-copulatory sexual selection. Significance of the StudySeminal fluid plays an important role in fertilisation success, yet the molecular components of seminal fluid and their function remain poorly understood in most species. This study provides the first comprehensive proteomic characterisation of a unique seminal foam produced by the Japanese quail (Coturnix japonica), revealing its potential mechanistic function in sperm regulation, sperm motility, immune protection, and fertilisation success. By identifying key proteins associated with energy production, membrane fluidity, and sperm maturation, our findings suggest the foam may influence post-ejaculatory sperm function. Additionally, the identification of immunity proteins indicates a role in sperm protection and modulation of female immune responses. This study advances our understanding of avian reproductive biology and opens new avenues for investigating the specific role of seminal foam proteins in fertilisation dynamics and post-copulatory sexual selection.

evolutionary biology↗

bioRxiv2025

Spatial and Temporal Patterns of Public Transit Aerobiomes

BackgroundAerobiome diversity is extensive, however community structure at the species level remains elusive. Likewise, microbiomes of public transit systems are of public interest due to their importance for health, though few studies have focused on these ecosystems whilst utilising shotgun metagenomics. Aerosol studies have focused predominantly on individual cities, with limited between-city comparisons suggesting specific community structures. Longitudinal studies show aerobiome diversity as dynamic, fluctuating during seasonal and daily cycles, though an annual cycle remains to be considered. Further, a bacterial bias has limited fungal aerobiome studies, with few considering both fractions collectively. As such, the objective of this study was to examine spatial and temporal patterns in the species diversity of public transit aerobiomes, with an emphasis on bacteria and fungi. ResultsAir samples taken over a three-year period (2017-2019) from six global cities were subjected to shotgun metagenomic sequencing. Improved classification databases, notably for fungi, applying stringent parameters for trimming, exogenous contamination removal and classification yielded high species-level resolution. Microbial diversity varied substantially among cities, while human and environmental factors, recorded in parallel, were of secondary significance. Bacteria dominated the public transit aerobiome with increased presence in cities with higher population densities. All aerobiomes had complex compositions, consisting of hundreds to thousands of species. Annual variation had limited significance on the public transit aerobiome diversity and community structure. ConclusionsCities were the most important factor contributing to diversity and community structure, demonstrating specific bacterial and fungal signatures. Further, a correlation between geographical distance and the genetic signatures of aerobiomes is suggested. Bacteria are the most abundant constituent of public transit aerobiomes, though no single species is globally dominant, conversely indicating a large inter-city variation in community structure. The presence of a ubiquitous global species core is rejected, though an aerobiome sub-core is confirmed. For the first time, local public transit aerobiome cores are presented for each city and related to ecological niches. Further, the importance of a robust bioinformatics analysis pipeline to identify and remove exogenous contaminants for studying low biomass samples is highlighted. Lastly, a core and sub-core definition of contaminant aerobiome species with taxon tables, to facilitate future environmental studies, is presented.

microbiology↗