Biology subjects

Fonseca, S.

Publications and source records attributed to Fonseca, S..

10 recordsLinked to original sources

bioRxiv2026

Barcoded-Plasmid DNA library construction for recording cell lineage trees enabled by a Scalable and modular Biofoundry-based Automated Robotic Pipeline

High-quality plasmid DNA purification at high throughput remains a significant bottleneck in molecular biology and bioengineering. Current methods frequently fail to deliver sufficient yields of pure, transfection-grade DNA required for genetic engineering applications in mammalian cells. Here, we present a Biofoundry-based automated pipeline using the CyBio FeliX robotic liquid handling platform to rapidly purify plasmid DNA with minimal manual intervention. The protocol leverages Solid Phase Reversible Immobilisation (SPRI)-based magnetic bead technology to ensure consistency, scalability, and DNA purity suitable for downstream viral particle production and mammalian cell transfection. The pipeline supports flexible processing of between 8 and 96 samples per run, making it adaptable across a wide range of experimental scales. The protocol is openly available via Earlham Institute GitHub repository, enabling broad adoption across the bioscientific community and contributing to the growing toolkit of reproducible, scalable engineering biology workflows. In this work, we employed an integrated robotic pipeline to process 528 pooled DNA plasmids and built a Lentiviral DNA plasmid library for lineage tracing, validated the library by sequencing, and demonstrated efficacy in downstream mammalian cell transfection experiments.

synthetic biology↗

bioRxiv2026

FlowFI: an interactive graphical software package for bespoke design of imaging parameters in flow cytometry to explore morphological diversity in bone marrow megakaryocytes

BackgroundImaging flow cytometry (IFC) provides a high quantity of single-cell morphological data, yet the field lacks open access tools for designing interpretable, bespoke parameters. In particular, rare and atypical cell populations where well annotated data is limited, are negatively affected. ResultsWe present Flow cytometry Feature Importance (FlowFI), an open-source graphical software for bespoke image parameter design and analysis. FlowFI provides a suite of image parameter options combining data across multiple channels and markers, tailored digital noise reduction (reducing noise resulting from common flow cytometry ultra-high image acquisition modalities), and a scalable, unsupervised feature selection pipeline that allows experimentalists to refine image-derived parameters iteratively, with a novel ensemble subsampling approach that provides robust feature importance scoring. We validated FlowFI using data from a rare and heterogenous bone marrow cell type, megakaryocytes, demonstrating that the tool can successfully identify novel, discriminatory morphological features to improve the purity of selected cell populations and gating strategy. ConclusionFlowFIs core functionalities are interacted with through an intuitive user interface for researchers with options to export data directly to common image and flow cytometry software formats. With this in mind, FlowFI offers a scalable way to both feature design, and feature refinement using a range of approaches to manifold learning, augmented by a data efficient bootstrap subsampling approach for unsupervised parameter recommendations in the big data regime. The software also introduces a new feature selection measures based on common manifold learning methods in the space inspired by the Uniform Manifold Approximation and Projection (UMAP) algorithm and finds performance comparable to existing methods. FlowFI provides a versatile testing ground for future developments in broad and dynamically developing areas of research including single cell analysis, label-free sorting and intra- and inter-cellular interaction analysis, while ensuring interoperability with current research workflows. Desktop installation options as well as detailed documentation can be found at https://github.com/EarlhamInst/FlowFI

cell biology↗

bioRxiv2024

The structure of the R2T complex reveals a different architecture of the related HSP90 co-chaperones R2T and R2TP

Heat shock protein 90 (HSP90) is a molecular chaperone that contributes to the maturation and activation of substrates in multiple cellular pathways. Its activity is supported by various co-chaperones. One of these is R2TP, a complex of RuvBL1-RuvBL2-RPAP3-PIH1D1 in humans, which is involved in the assembly of various multiprotein complexes, including mTORC1 and Box C/D and Box H/ACA snoRNPs. Structural analyses have shown that the complex is organized around a heterohexameric ring of the ATPases RuvBL1-RuvBL2 in both yeast and humans. In addition, several R2TP-like co-chaperones have been identified in humans, such as R2T, which lacks PIH1D1, but these are less well characterized. In seed plants, there are no PIH1D1 orthologs. Here, we have identified the R2T complex of Arabidopsis and determined its cryoEM structure. R2T associates with the prefoldin-like complex in vivo and is located in the cytosolic and nuclear compartments. R2T is organized as a dodecamer of AtRuvBL1-AtRuvBL2a that forms two rings, with one AtRPAP3 anchored to each ring. AtRPAP3 has no effect on the ATPase activity of AtRuvBL1-AtRuvBL2a and binds with a different stoichiometry than that described for human R2TP. We show the interaction of AtRPAP3 with AtRuvBL2a and AtHSP90 in vivo and describe the residues involved. Taken together, our results show that AtRPAP3 recruits AtRuvBL1-AtRuvBL2a and AtHSP90 via a mechanism that is also conserved in other eukaryotes, but that R2T and R2TP co-chaperone complexes have distinct structures that also suggest differences in their functions and mechanisms.

biochemistry↗

bioRxiv2024

An active light signalling pathway is necessary for ABA-induced inhibition of hypocotyl elongation.

Driven by cell elongation, hypocotyl growth is tightly controlled by light and responds to external stimuli and endogenous hormonal pathways. Hypocotyls are known to be responsive to the stress signalling hormone abscisic acid (ABA) which effectively inhibits cell elongation, but how this regulation is connected to light responses and other endogenous hormonal pathways has been a subject of limited studies. Here, we show that whereas hypocotyl elongation is sensitive to ABA in light-grown seedlings, the hypocotyl of dark-grown etiolated seedlings is ABA-insensitive. In the dark, hypocotyl sensitivity to ABA is restored in the constitutive photomorphogenic pifq and cop1-4 mutants, suggesting that an active light signalling pathway is necessary for hypocotyl responsiveness to ABA. However, etiolated hypocotyls retain ABA responsiveness, as could be detected by the induction of ABI1 and RD29B transcripts in response to exogenous ABA, suggesting that inhibition of hypocotyl elongation mediated by ABA does not follows the canonical ABA signalling dependent on transcription. Here, using RNA-seq analysis we identified a number of ABA differentially expressed genes (DEGs) that correlate with ABA inhibition of hypocotyl elongation, specifically in dark-grown pifq or light-grown WT plants, and whose expression remains unchanged by ABA treatment in dark-grown WT plants. Among these DEGs we identified a number of genes playing a role in cell elongation directly at the level of the plasma membrane, as SAURs, ion transporters, auxin flux regulators, channels, and cell wall modification enzymes. The use of the auxin transport inhibitor, NPA, revealed that in the light auxin transport impairment renders hypocotyls insensitive to ABA in WT and pifq plants. Thus, in the light, hypocotyl responsiveness to ABA is dependent on auxin transport and independent of PIFs. In the dark, PIFs render hypocotyls insensitive to ABA, perhaps by regulating the expression of a number of ABA DEGs, a mechanism that could allow plants to prioritize the elongation towards light, avoiding to slow-down soil emergence that could be induced by ABA signalling in case of sudden reduction of soil moisture.

plant biology↗

bioRxiv2021

COP1 promotes seed germination by destabilizing RGA-LIKE2 (RGL2) in Arabidopsis

Under favorable moisture, temperature and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGL2, a major repressor of germination in Arabidopsis seeds. Analysis of seed germination phenotypes of constitutively photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacts directly with RGL2 and in vivo this interaction is strongly enhanced by SPA1. COP1 directly ubiquitinates RGL2 to promote its degradation. Moreover, GA stabilizes COP1 with consequent RGL2 destabilization. By uncovering this COP1-RGL2 regulatory module, we reveal a novel mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

plant biology↗

bioRxiv2021

Secreted SARS-CoV-2 ORF8 modulates the cytokine expression profile of human macrophages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still adapting to its new human host. Attention has focussed on the viral spike protein, but substantial variation has been seen in the ORF8 gene. Here, we show that SARS-CoV-2 ORF8 protein undergoes signal peptide-mediated processing through the endoplasmic reticulum and is secreted as a glycosylated, disulphide-linked dimer. The secreted protein from the prototype SARS-CoV-2 virus had no major effect on viability of a variety of cell types, or on IFN or NF-{kappa}B signalling. However, it modulated cytokine expression from primary CSF1-derived human macrophages, most notably by decreasing IL-6 and IL-8 secretion. Furthermore, a sequence polymorphism L84S that appeared early in the pandemic associated with the Clade S lineage of virus, showed a markedly different effect, of increasing IL-6 production. We conclude that ORF8 sequence polymorphisms can potentially affect SARS-CoV-2 virulence and should therefore be monitored in sequencing-based surveillance.

microbiology↗

bioRxiv2021

Extracellular vesicles produced by the human commensal gut bacterium Bacteroides thetaiotaomicron affect host immune pathways in a cell-type specific manner that are altered in inflammatory bowel disease

The gastrointestinal (GI) tract is inhabited by a complex microbial community, which contributes to its homeostasis. Disrupted microbiome can cause GI-related diseases, including inflammatory bowel disease, therefore identifying host-microbe interactions is crucial for better understanding gut health. Bacterial extracellular vesicles (BEVs), released into the gut lumen, can cross the mucus layer and access underlying immune cells. To study cross-kingdom communication between BEVs and host, we focused on the influence of BEVs, generated by Bacteroides thetaiotaomicron (VPI-5482), on host immune cells. Using single-cell RNA sequencing data and host-microbe protein-protein interaction networks, we examined the potential effect of BEVs on dendritic cells, macrophages and monocytes with particular focus on the Toll-like receptor (TLR) pathway. We identified biological processes affected in each immune cell type, and also cell-type specific processes (e.g myeloid cell differentiation). The TLR pathway analysis highlighted that BEV targets differ among cells and even between the same cells in healthy versus disease (ulcerative colitis) conditions. Our in silico findings were validated in BEV-monocyte co-cultures demonstrating the requirement for TLR4 in BEV-elicited NF-{kappa} B activation. This study demonstrates that both cell-type and health condition influence BEV-host communication. The results and the pipeline can facilitate BEV-based therapy development for the treatment of IBD.

bioinformatics↗

bioRxiv2021

Regulation of blood-brain barrier integrity and cognition by the microbiome-associated methylamines trimethylamine N-oxide and trimethylamine

BackgroundCommunication between the gut microbiota and the brain is primarily mediated via soluble microbe-derived metabolites, but the details of this pathway remain poorly defined. Methylamines produced by microbial metabolism of dietary choline and L-carnitine have received attention due to their proposed association with vascular disease, but their effects upon the cerebrovascular circulation have hitherto not been studied. ResultsHere we use an integrated in vitro/in vivo approach to show that physiologically relevant concentrations of the dietary methylamine trimethylamine N-oxide (TMAO) enhanced blood-brain barrier (BBB) integrity and protected it from inflammatory insult, acting through the tight junction regulator annexin A1. In contrast, the TMAO precursor trimethylamine (TMA) impaired BBB function and disrupted tight junction integrity. Moreover, we show that long-term exposure to TMAO protects murine cognitive function from inflammatory challenge, acting to limit astrocyte and microglial reactivity in a brain region-specific manner. ConclusionOur findings demonstrate the mechanisms through which microbiome-associated methylamines directly interact with the mammalian BBB, with consequences for cerebrovascular and cognitive function.

systems biology↗

bioRxiv2020

DET1-mediated COP1 regulation avoids HY5 activity over second-site targets to tune plant photomorphogenesis

DE-ETIOLATED1 (DET1) is a negative regulator of plant photomorphogenesis acting as a component of the C3D complex, which can further associate to CULLIN4 to form a CRL4C3D E3 ubiquitin ligase. CRL4C3D is thought to act together with CRL4COP1SPA ubiquitin ligase, to promote the ubiquitin-mediated degradation of the master regulatory transcription factor ELONGATED HYPOCOTYL5 (HY5), thereby controlling photomorphogenic gene regulatory networks. Yet, functional links between COP1 and DET1 have long remained elusive. Here, upon mass spectrometry identification of DET1 and COP1-associated proteins, we provide in vivo evidence that DET1 associates with COP1 to promote its destabilization, a process necessary to dampen HY5 protein abundance. By regulating HY5 over-accumulation, DET1 is critical to avoid its association to second-site loci, including many PIF3 target genes. Accordingly, excessive HY5 levels result in an increased HY5 repressive activity and are sufficient to trigger fusca-like phenotypes otherwise observed typically in COP1 and COP9 signalosome mutant seedlings. This study therefore identifies that DET1-mediated regulation of COP1 stability tunes down HY5 cistrome and avoids hyper-photomorphogenic responses that might compromise plant viability.

plant biology↗

bioRxiv2018

DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a plant deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

plant biology↗