Biology subjects

Masek, J.

Publications and source records attributed to Masek, J..

7 recordsLinked to original sources

bioRxiv2026

Cyanochelin uptake reveals an exclusively cyanobacterial class of AMIN-domain TonB-dependent transporters

Siderophore transport is central to microbial competition, because it determines access to iron, frequently a limiting nutrient. While siderophore-mediated iron uptake via TonB-dependent transporters (TBDTs) has been extensively studied in heterotrophic bacteria, little is known about the functionality and specificity of TBDTs in cyanobacteria. In the present study we functionally characterise the import system of cyanochelin B, a photolytic {beta}-hydroxy aspartate siderophore produced by several filamentous cyanobacteria, including Leptolyngbya sp. NIES-3755. We have identified a cyanochelin B putative transport cassete localized in the vicinity of the cyanochelin biosynthetic gene cluster in Leptolyngbya genome. By expressing the import genes heterologously in a model unicellular cyanobacterium Synechocystis sp. PCC 6803, we established that the transport cassette reconstitutes cyanochelin B-dependent growth, consistent with cyanochelin-mediated iron acquisition. Systematic gene dissection showed that the TBDT (CctA) and the substrate-binding protein (CctB), responsible for binding the siderophore in the periplasm, are alone sufficient for cyanochelin import into Synechocystis cells, with the permeases, ATPase and a cassette-associated ferredoxin supplied in trans by the host. CctA carries an N-terminal AMIN domain, a fusion found only in cyanobacterial TBDTs. The cassette accepts the structurally similar cyanochelin A but not cyanochelin C, enterobactin or pyoverdine, indicating limited promiscuity. The phylogenetic placement of cyanochelin receptors within a broader clade containing citrate-hydroxamate-type siderophore receptors suggests an evolutionary link between transport systems for chemically distinct cyanobacterial siderophores. Our study reports the first functional heterologous expression of a cyanobacterial TonB-dependent transporter and establishes Synechocystis as a promising platform for cyanobacterial xenosiderophore-uptake studies.

microbiology↗

bioRxiv2026

Utilization of cyanobacterial siderophore cyanochelin B by phylogenetically distant heterotrophs suggest its role in mediating microbial interactions

Cyanobacteria are key prokaryotic primary producers in diverse ecosystems, yet the role of cyanobacterial siderophores in shaping their associated microbiomes remains unexplored. Our study demonstrates the benefits provided to the heterotrophic co-habitants of filamentous cyanobacteria in terrestrial microbial biofilms, focusing on the recently discovered widespread siderophores cyanochelins. To address the acceptance of cyanochelin B (CychB) across multiple bacterial classes, we first investigated its role in providing iron to a model siderophore producer P. aeruginosa PAO1 and selected Pseudomonas natural isolates, which were found to utilize CychB under iron limiting conditions while downregulating endogenous siderophore production. In response to CychB, PAO1 expresses a siderophore internalization cluster, which is localized in multiple Pseudomonas natural isolates. Using metagenome analysis, we characterized the bacterial community recruited along with CychB producing Phormidesmis cyanobacteria under long-term iron starvation. Potential CychB acceptor bacteria associated with the CychB producer were predominantly lacking endogenous siderophore machineries. Using siderophore selective pressure, we isolated a genuine CychB acceptor, gram-negative bacterium Methyloversatilis sp. S146 and demonstrated that its genome hosts an iron processing cluster overexpressed after CychB feeding, recognizing Methyloversatilis as a candidate for further mechanistic investigation of iron acquisition-driven microbial interactions. Our results indicate that CychB supports a specific subset of co-habiting heterotrophic bacteria during iron starvation, further emphasizing the role of cyanobacteria as key drivers of nutrient flows within globally important microbial soil crust ecosystems, supporting microbial life in nutrient-limited environments. These findings provide a mechanistic foundation to elucidate the role of cyanochelins as a public good in these communities.

microbiology↗

bioRxiv2026

NOTCH1-specific phosphorylation of S1970 by Casein Kinase 1 is required for NOTCH1 transcriptional competence and signaling activity in vivo

The Notch and Wnt/{beta}-catenin signaling pathways are essential regulators for cell-fate decisions, cellular patterning, and tissue homeostasis. Multiple studies point to their orchestrated role during development, but the molecular mechanism of the protein-protein crosstalk is largely unknown. Here, after screening effects of Wnt/{beta}-catenin component loss on NOTCH1 protein, we identify Casein Kinase 1 (CK1) as a positive regulator of NOTCH1 activity in vitro and in vivo. We demonstrate that CK1 associates with NOTCH1 and that its kinase activity is required to sustain Notch-driven transcription. Using UltraID proximity-assay, we revealed that CK1 is required for the NOTCH1 interactivity with transporter proteins, and MAML1 both prior and after ligand-induced activation. Combining structural modelling, NMR, and mass spectrometry, we identified Serine 1970 (S1970) as a previously unreported residue within the Notch1 Intracellular Domain (N1ICD) essential for its signaling competence. Our modeling predicts that the phosphorylation of S1970 facilitates an intra-domain conformational switch with R1937 and R1962 residues altering the assembly of the N1ICD-MAML1-RBPJk transcriptional complex. Finally, we demonstrate the biological significance of N1ICD S1970 in vivo using Xenopus laevis axis-duplication rescue assay. Our results establish CK1 as a key positive mediator of the Notch receptor transcriptional activity.

cell biology↗

bioRxiv2024

The ReAct project: Analysis of data from 23 differentlaboratories to characterise DNA recovery given twosets of activity level propositions

The ReAct (Recovery, Activity) project is an ENFSI (European Network of Forensic Science Institutes) supported initiative comprising a large consortium of laboratories. Here, the results from more than 23 laboratories are presented. The primary purpose was to design experiments simulating typical casework circumstances; collect data and to implement Bayesian networks to assess the value (i.e., likelihood ratio) of DNA results given activity level propositions. Two different experimental designs were used to simulate a robbery, where a screwdriver was used to force a door or window. Propositions and case information were chosen following laboratory feedback listing typical casework circumstances (included in the paper). Whereas the proposition representing prosecutions view is applicable to many cases: the defendant forced the door/window with his screwdriver; the proposition representing defences view are more variable depending on what is alleged to have happened. In a direct transfer experiment, the defendant owned and used the screwdriver, but he did not force the door/window in question. An unknown person used the defendants stolen screwdriver. In an indirect transfer experiment, the defendant neither owned, saw, nor used the screwdriver, nor did they force the door or window. Instead, an unknown individual--someone with whom the defendant had interacted (e.g., by shaking hands or touching the same objects)--used the recovered tool to force entry. In the first experiment, if the defendant forced the door/window, then s/he is the last handler of the screw driver, whereas if the tool was stolen and used by an unknown person, the defendant is the first handler. For the second experiment, given the defence view, the defendant never held the screwdriver. We envisaged the situation where an object manipulated by the defendant (or the defendant himself/herself) would be touched by the unknown offender who would then force the window. The time delay between the touching of the object and the forcing of the window was 0, 1h, and 2h. This was accomplished by handshaking experiments, where the person forcing the door would shake a persons hand, and then use the screwdriver either immediately after hand-shake or after 1, 2 hours. It was found for the direct transfer experiment that unless a single contributor profile aligning with the known persons of interest profile was retrieved, the results did not allow to discriminate propositions. On the other hand, for the indirect transfer experiment, both single and major contributor profiles that aligned with the person of interest (POI) supported the proposition that the person used the tool rather than an unknown person who had touched an object, when indeed the former was true. There was considerable variation in median recoveries of DNA between laboratories (between 200pg - 5ng) for a given experiment if quantities are taken into account. These differences affect the likelihood ratios given activity level propositions. More than 2,700 samples were analysed in the course of this study. Two different Bayesian Networks are made available via an open source application written in Shiny R: Shiny_React(). For comparison, all datasets were analysed using a qualitative method categorised into absent, single, balanced or major given contributors. The need for standardisation of methods is highlighted, along with the need for new methods to assess probability of laboratory dependent DNA recovery. Open access databases that are made freely available are important adjuncts

genetics↗

bioRxiv2022

Protection from liver cancer in a mouse model of Alagille syndrome follows dysregulated differentiation of thymocytes and hepatocytes

Fibrosis is a physiological tissue repair mechanism, but excessive fibrosis can disrupt organ function. Alagille syndrome (ALGS), which is caused by mutations in the Notch ligand JAGGED1, results in bile duct paucity, neonatal cholestasis, and a characteristic fibrotic response. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulates ALGS-like pericellular fibrosis. Single-cell RNA-seq and multi-color flow cytometry characterization of the liver and spleen revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration in cholestatic Jag1Ndr/Ndr mice, despite an enrichment in extrahepatic (thymic and splenic) regulatory T cells (Tregs). Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive immune cell transplantation into Rag1-/- mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes, in response to DSS. Cholestasis induced by BDL in Rag1-/- mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1-/- mice with Jag1+/+ lymphocytes. Finally, we show that the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation are corroborated by transcriptomic data from children with ALGS. In sum, these data lead to a model in which Jag1-driven developmental hepatic and immune defects interact to determine the fibrotic process in ALGS.

developmental biology↗

bioRxiv2021

Spatial Transcriptomics to define transcriptional patterns of zonation and structural components in the liver

Reconstruction of heterogeneity through single-cell transcriptional profiling has greatly advanced our understanding of the spatial liver transcriptome in recent years. However, global transcriptional differences across lobular units remain elusive in physical space. Here, we implement Spatial Transcriptomics to perform transcriptomic analysis across sectioned liver tissue. We confirm that the heterogeneity in this complex tissue is predominantly determined by lobular zonation. By introducing novel computational approaches, we enable transcriptional gradient measurements between tissue structures, including several lobules in a variety of orientations. Further, our data suggests the presence of previously transcriptionally uncharacterized structures within liver tissue, contributing to the overall spatial heterogeneity of the organ. This study demonstrates how comprehensive spatial transcriptomic technologies can be used to delineate extensive spatial gene expression patterns in the liver, indicating its future impact for studies of liver function, development and regeneration as well as its potential in pre-clinical and clinical pathology.

genomics↗

bioRxiv2020

Neural Plate Targeting by in Utero NanoinjEction (NEPTUNE) reveals a role for Sptbn2 in neurulation and abdominal wall closure

Gene variants associated with disease are efficiently identified with whole genome sequencing or GWAS, but validation in vivo lags behind. We developed NEPTUNE (neural plate targeting by in utero nanoinjection), to rapidly and flexibly introduce gene expression-modifying viruses to the embryonic murine neural plate prior to neurulation, to target the future adult nervous system. Stable integration in >95% of cells in the brain enabled long-term gain- or loss-of-function, and conditional expression was achieved using mini-promotors for cell types of interest. Using NEPTUNE, we silenced Sptbn2, a gene associated with Spinocerebellar ataxia type 5 (SCA5) in humans. Silencing of Sptbn2 induced severe neural tube defects and embryo resorption, suggesting that SPTBN2 in-frame and missense deletions in SCA5 reflect hypomorphic or neomorphic functions, not loss of function. In conclusion, NEPTUNE offers a novel, rapid and cost-effective technique to test gene function in brain development, and can reveal loss of function phenotypes incompatible with life.

developmental biology↗