Biology subjects

Bloomfield, M.

Publications and source records attributed to Bloomfield, M..

8 recordsLinked to original sources

bioRxiv2026

Comprehensive Profiling of Age- and Immune Cell- Specific Signaling Activation Using Multiplex Phosphoflow

Immune phenotyping represents a pillar in diagnostics, characterization of new genetic defects, and understanding mechanisms of diseases. Cell population distribution often does not cover the intrinsic function changes that may contribute to disease. Outcome of signaling activation can be used as proxy for cell function. To overcome the limitation of sample availability and standardization of signaling assays, we developed a multiplex full spectrum cytometry phosphoflow assay allowing the study of 6 phospho-proteins representing BCR/TCR, MAPK, PI3K/Akt/mTOR and canonical NF-{kappa}B signaling pathways in 18 immune cell subpopulations. Maximal stimulation and temporal dynamics were studied in response to pan-stimuli, activating cells regardless of receptor, and targeted stimuli for T, B, and innate immune cells. We studied healthy individuals between 1-69 years and discovered subpopulations-specific responses. Furthermore, pediatric donors showed broad differences in B cell and T cell function compared to adults. Hence, we established a tool to assess multiple signaling pathways at once and provide age- and subpopulation-specific references for signaling outcome. SummaryMultiplex full spectrum flow cytometry-based phosphoflow assay across 18 immune cell subpopulations, 6 phospho-proteins in response to 6 stimuli at 4 time points in individuals aged 1-69 years, reveals distinct age- and subpopulation-associated signaling patterns in magnitude and dynamics of pathways activation.

immunology↗

bioRxiv2026

Co-infections and cryptic pathogens uncovered by metatranscriptomics in New Zealands severe acute respiratory infections

Severe acute respiratory infections (SARI) are a leading cause of hospitalisation and mortality globally. Many SARI cases remain undiagnosed because kit-based PCR diagnostic panels are typically limited to one or a small number of known pathogens and may fail to identify low-abundance infections or novel, poorly characterised organisms. Here, we used metatranscriptomic sequencing to profile the total infectome of 300 PCR-negative SARI nasopharyngeal samples collected through sentinel hospital-based surveillance in New Zealand between 2014-2021. Our analysis revealed actively transcribing potential pathogens in 43% of SARI cases, comprising 10 RNA viruses, three DNA viruses, nine bacterial species and four fungal species. Notably, co-infections occurred in 26% of cases, revealing polymicrobial infections missed by routine diagnostics. Human rhinoviruses were the most frequently identified, despite not being detected by PCR, and multiple common-cold coronaviruses, human parechovirus A1 and parainfluenza virus type 4, were identified, although these were not included in the PCR screening panel. We also detected a range of bacterial and fungal species and uncovered highly expressed virulence and antimicrobial resistance genes. Infectome composition and diversity were shaped by key demographic and epidemiological factors, with strongest effects observed for age and year of sample collection, indicating that host characteristics and temporal dynamics influence both microbial richness and community structure. These findings highlight the limitations of current diagnostic strategies and the value of metatranscriptomics for comprehensive microbial identification. Integrating such genomic approaches into both clinical and public health frameworks could improve diagnostic accuracy, enabling more sensitive detection and characterisation of potential pathogens while also strengthening surveillance and outbreak response.

genomics↗

bioRxiv2025

Cell and nuclear size are associated with chromosomal instability and tumorigenicity in cancer cells that undergo whole genome doubling

Whole genome doubling (WGD) is a frequent event in cancer evolution associated with chromosomal instability, metastasis, and poor prognosis. While the genomic consequences of WGD are well documented, the effects of non-genetic alterations that accompany WGD, such as changes to cell and nuclear size, on tetraploid (4N) cancer cell physiology are less understood. Here, we show that cell and nuclear volume do not always scale with DNA content after WGD in cancer cells, resulting in 4N cells that differ in size. We find that small size is associated with enhanced cell fitness, mitotic fidelity, and tumorigenicity in 4N cancer cells and with poor patient survival in WGD-positive human cancers. Overall, these results suggest that cell and nuclear size contribute to the tumorigenic potential of 4N cancer cells and could be an important prognostic marker in human tumors that undergo WGD. Statement of SignificanceWe report that WGD generates tetraploid cancer cells that vary in size, with larger cells displaying high chromosomal instability and smaller cells exhibiting high fitness and tumorigenicity. Furthermore, WGD status and cancer cell nuclear size in human tumors correlated with patient survival, demonstrating the clinical relevance of this association.

cancer biology↗

bioRxiv2024

Resolving a neonatal intensive care unit outbreak of methicillin-resistant Staphylococcus aureus to the SNV level using Oxford Nanopore simplex reads and HERRO error correction

ObjectivesOur laboratory began prospective genomic surveillance for healthcare-associated organisms in 2022 using Oxford Nanopore Technologies (ONT) sequencing as a standalone platform. This has permitted the early detection of outbreaks but has been insufficient for single-nucleotide variant (SNV)-level analysis due to lower read accuracy than Illumina sequencing. This study aimed to determine whether Haplotype-aware ERRor cOrrection (HERRO) of ONT data could permit high-resolution comparison of outbreak isolates. MethodsWe used ONT simplex reads from isolates involved in a recent outbreak of methicillin-resistant Staphylococcus aureus (MRSA) in our neonatal unit. The raw sequence data were re-basecalled and adapter-trimmed using Dorado v0.7.0. The simplex reads then underwent HERRO correction. The resulting genome assemblies and phylogenies were compared with previous analyses (using Dorado v0.3.4, no HERRO correction and data generated by Illumina sequencing). ResultsFive of nine outbreak isolates were included in the analysis. The remaining four isolates had insufficient read lengths (N50 values <10,000 bp) and did not provide complete chromosome coverage after HERRO correction. The average chromosome sequencing depth for nanopore data was 147x (range: 44-220x) with an average read N50 of 12,215 bp (interquartile range (IQR): 11,439-12,711 bp). The median pairwise SNV distance between outbreak isolates from the original investigation was 51 SNVs (range: 40-68), which decreased to 3 SNVs (range: 1-15) with HERRO correction. Illumina sequencing generated a median SNV distance of 2 (range: 0-13). The resulting standalone ONT HERRO-corrected phylogeny was almost indistinguishable from the standalone Illumina-generated phylogeny. ConclusionsThe addition of HERRO correction meant isolates from this MRSA outbreak could be resolved to a level on par with Illumina sequencing. ONT data following HERRO correction represents a viable standalone option for high-resolution genomic analysis of hospital outbreaks, provided sufficient read lengths can be generated.

genomics↗

bioRxiv2022

Digital data repository and automatic analysis framework for FDOPA PET neuroimaging

IntroductionFDOPA PET has been used extensively to image the human brain in many clinical disorders and has the potential to be used for patient stratification and individualized treatment. However, to reach its full and effective clinical translation, FDOPA PET requires both a robust data infrastructure and analytical protocol that are capable of ensuring high quality data and metadata, accurate biological quantification, and replicable results. In this study we evaluate a digital data repository and automated analytical framework for FDOPA PET neuroimaging that can produce an individualised quantification of dopamine synthesis capacity in the living human brain. MethodsThe imaging platform XNAT was used to store the Kings College London institutional brain FDOPA PET imaging archive, alongside individual demographics and clinical information. A fully automated analysis pipeline for imaging processing and data quantification was developed in Python and integrated in XNAT using the Docker technology. Reproducibility was assessed in test-retest datasets both in controls and patients with psychosis. The agreement between the automated analysis estimates and the results derived by the manual analysis were compared. Finally, using a sample of healthy controls (N=115), a sensitivity analysis was performed to explore the impact of experimental and demographic variables on the FDOPA PET measures. ResultsThe final data repository includes 892 FDOPA PET scans organized from 23 different studies, collected at five different imaging sites. After removing commercials studies, the infrastructure consisted of 792 FDOPA PET scans from 666 individuals (female 33.9%, healthy controls 29.1%) collected from four different imaging sites between 2004-2021. The automated analysis pipeline provided results that were in agreement with the results from the manual analysis, with a Pearsons correlation that ranged from 0.64 to 0.99 for Kicer, and from 0.79 to 1.00 for SUVR. The mean absolute difference between the two pipelines ranges from 3.4% to 9.4% for Kicer, and from 2.5% to 12.4% for SUVR. Moreover, we found good reproducibility of the data analysis by the automated pipeline (in the whole striatum for the Kicer: ICC for the controls = 0.71, ICC for the psychotic patients = 0.88). From the demographic and experimental variables assessed, gender was found to most influence striatal dopamine synthesis capacity (F = 10.7, p <0.001), with women showing greater dopamine synthesis capacity than men, while the effects of weight, age, injected radioactivity, and scanner, varied by brain region and parameter of interest. ConclusionsCombining information from different neuroimaging studies has allowed us to test comprehensively the automated pipeline for quantification of dopamine synthesis capacity using FDOPA PET data and to validate its replicability and reproducibility performances on a large sample size. This validation process is a necessary methodological step for the development of the clinical application of FDOPA PET as precision medicine biomarker. The proposed infrastructure is generalisable behind the FDOPA radiotracer.

bioengineering↗

bioRxiv2021

Whole-genome duplication shapes the aneuploidy landscape of human cancers

Aneuploidy - a hallmark of cancer - has tissue-specific recurrence patterns suggesting it plays a driving role in cancer initiation and progression. However, the contribution of aneuploidy to tumorigenesis depends on the cellular and genomic context in which it arises. Whole-genome duplication (WGD) is a common macro-evolutionary event that occurs in >25% of human tumors during the early stages of tumorigenesis. Although tumors that have undergone WGD are reported to be more permissive to aneuploidy than tumors that have not, it remains unknown whether WGD affects aneuploidy recurrence patterns in human cancers. Here we analyzed clinical tumor samples from 449 WGD- tumors and 157 WGD+ tumors across 22 tumor types. We found distinct recurrence patterns of aneuploidy in WGD- and WGD+ tumors. The relative prevalence of recurrent aneuploidies decreased in WGD+ tumors, in line with increased aneuploidy tolerance. Moreover, the genetic interactions between chromosome arms differed between WGD- and WGD+ tumors, giving rise to distinct co-occurrence and mutual exclusivity aneuploidy patterns. The proportion of whole-chromosome aneuploidy vs. arm-level aneuploidy was significantly higher in WGD+ tumors, indicating distinct dominant mechanisms for aneuploidy formation in WGD- and WGD+ tumors. Human cancer cell lines successfully reproduced these WGD/aneuploidy interactions, confirming the relevance of studying this phenomenon in culture. Lastly, we induced WGD in human colon cancer cell lines, and followed aneuploidy formation in the isogenic WGD+/WGD-cells under standard or selective conditions. These experiments validated key findings from the clinical tumor analysis, and revealed a causal link between WGD and altered aneuploidy landscapes. We conclude that WGD shapes the aneuploidy landscape of human tumors, and propose that the interaction between WGD and aneuploidy is a major contributor to tumor evolution.

cancer biology↗

bioRxiv2020

Individual and combined effects of Cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC) on striato-cortical connectivity in the human brain

Cannabidiol (CBD) and {Delta}9-tetrahydrocannabinol (THC) are two major constituents of cannabis with contrasting mechanisms of action. THC is the major psychoactive, addiction-promoting, and psychotomimetic compound, while CBD may have somewhat opposite effects. The brain effects of these drugs alone and in combination are poorly understood. In particular the striatum is implicated in the pathophysiology of several psychiatric disorders, but it is unclear how THC and CBD influence striato-cortical connectivity. Across two placebo-controlled, double-blind studies, we examine the effects of THC, CBD, and THC+CBD on the functional connectivity of striatal sub-divisions (associative, limbic, and sensorimotor) using resting-state functional Magnetic Resonance Imaging (fMRI) and seed-based functional connectivity analyses. Study 1 (N=17; inhaled 8mg THC, 8mg THC+10mg CBD, placebo) showed strong disruptive effects of both THC and THC+CBD conditions on connectivity in the associative and sensorimotor networks, but a specific effect of THC in the limbic striatum, which was alleviated in the THC+CBD condition such that it did not differ from placebo. In Study 2 (N=23, oral 600mg CBD, placebo) CBD increased connectivity in the associative network, but relatively minor decreases/disruptions were found in the limbic and sensorimotor. In conclusion, THC strongly disrupts striato-cortical networks, and this effect is selectively mitigated in the limbic striatum when co-administered with CBD. When administered alone, 600mg oral CBD has a more complex effect profile of relative increases and decreases in connectivity. The insula emerges as a key region affected by cannabinoid-induced changes in functional connectivity, with potential implications for understanding cannabis related disorders, and the development of cannabinoid therapeutics.

neuroscience↗

bioRxiv2020

A patient-designed tissue-engineered model of the infiltrative glioblastoma microenvironment

Glioblastoma is an aggressive brain cancer characterized by diffuse infiltration. Infiltrated glioma cells persist in the brain post-resection where they interact with glial cells and experience interstitial fluid flow. We recreate this infiltrative microenvironment in vitro based on resected patient tumors and examine malignancy metrics (invasion, proliferation, and stemness) in the context of cellular and biophysical factors and therapies. Our 3D tissue-engineered model comprises patient-derived glioma stem cells, human astrocytes and microglia, and interstitial fluid flow. We found flow contributes to all outcomes across seven patient-derived lines, and glial effects are driven by CCL2 and differential glial activation. We conducted a six-drug screen using four outcomes and find expression of putative stemness marker CD71, opposed to viability IC50, significantly predicts murine xenograft survival. Our results dispute the paradigm of viability as predictive of drug efficacy. We posit this patient-centric, infiltrative tumor model is a novel advance towards translational personalized medicine.

bioengineering↗