Biology subjects

Jones, M. C.

Publications and source records attributed to Jones, M. C..

11 recordsLinked to original sources

bioRxiv2025

VSA-2, a novel plant-derived adjuvant for SARS-CoV-2 subunit vaccine

QS-21, a key component of several licensed vaccines is facing limited supply, dose-limiting toxicity and other drawbacks which together limit its broader usage. Development of saponin alternatives to QS-21 that retain its desirable adjuvant activity without its drawbacks is in high need. Incorporating an amide side chain into the more sustainable Momordica saponins (MS) I and II led to the recent discovery of two semisynthetic immunostimulatory adjuvants VSA-1 and VSA-2. Here, we showed that the receptor-binding protein (RBD) of ancestral SARS-CoV-2 adjuvanted with VSA-2 (VSA-2-RBD) induced high titers of SARS-CoV-2-specific humoral and T helper-1 prone immune responses in mice comparable to that triggered by QS-21-RBD. Vaccination with VSA-2-RBD provided strong protection against SARS-COV-2, Delta and Omicron variants infection and the virus-induced lung inflammation and pathology similarly as QS-21-RBD vaccination. Overall, our results suggest that VSA-2 adjuvant can potentially complement the clinically proven saponin adjuvant QS-21 in vaccines against infectious diseases.

microbiology↗

bioRxiv2025

Development of a High-Throughput Ion Mobility Spectrometry-Based Screening Platform for Kainoid Synthase Biocatalysts

Random mutagenesis generates large, diverse enzyme libraries, however high-throughput screening is challenging when transformations yield isomeric small molecule products. We developed a matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry (MALDI-TIMS-MS) platform for rapid, label-free screening of libraries of kainoid synthases, Fe/KG-dependent cyclases that produce the isomeric neurotransmitters kainic acid (KA) and kainic acid lactone (KAL). This platform achieves near-baseline separation of KA and KAL directly from microbial biomass, with parallel reaction monitoring-PASEF (PRM-PASEF) enhancing signal-to-noise for isomeric product ratio analysis. Using this workflow, we screened a 318-variant DsKabC/GfKabC DNA shuffle library in under 30 minutes, identifying seven variants with improved KAL conversion. Two variants showed a near-complete shift toward KAL production with greater substrate consumption, while retaining favorable expression profiles compared to GfKabC. These results establish MALDI-TIMS-MS as a generalizable platform for high-throughput isomeric product screening and provides new kainoid synthase variants to probe structure-function relationships for further development as biocatalysts. Importantly, this screening approach can be broadly applied to interrogate enzyme variants acting on small molecules, delivering rapid and accurate quantitative insights without reliance on chromatography, enzyme purification, or substrate derivatization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/674781v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@185877dorg.highwire.dtl.DTLVardef@18234d6org.highwire.dtl.DTLVardef@15b0df9org.highwire.dtl.DTLVardef@f28f8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Respiratory syncytial virus infection confers heterologous protection against SARS-CoV-2 via induction of γδ T cell-mediated trained immunity and SARS-CoV-2 reactive mucosal T cells

The respiratory viruses can concurrently or sequentially infect a host and influence the trajectory of each other. The underlying immune mechanisms are not well understood. Here, we investigated whether respiratory syncytial virus (RSV) infection affects host vulnerability to subsequent SARS-CoV-2 infection in two murine models of SARS-CoV-2 infection. We found that prior RSV infection-induced heterologous protection against subsequent SARS-CoV-2 infection was dose and time dependent. RNA-seq and immunological analyses revealed that RSV triggered the activation of lung antigen presenting cells (APC)s and SARS-CoV-2 reactive mucosal T cells at day 9, which declined at 1 month. RSV also induced the expansion of lung {gamma}{delta} T cells and the upregulation of their cellular metabolic pathways. Furthermore, RSV infection in TCR{delta}-/- mice, which are deficient of {gamma}{delta} T cells, resulted in a reduced SARS-CoV-2 reactive mucosal T cell response and subsequent increased viral loads and higher levels of virus-induced inflammatory responses in the lung upon SARS-CoV-2 challenge compared to the wild-type mice. In summary, our findings suggest that RSV infection provides heterologous protection against the subsequent SARS-CoV-2 infection via induction of {gamma}{delta} T cell-mediated trained immunity in the lung and SARS-CoV-2 reactive mucosal T cell responses.

microbiology↗

bioRxiv2024

Differential roles of cyclin-CDK1 complexes in cell migration and invasion

Two key hallmarks of cancer are dysregulated proliferation and metastasis, which are co- ordinated at the single cell level by regulation of cell-cycle progression and invasive cell migration respectively. We have previously described a central role for CDK1 at the nexus of adhesion signalling and cell cycle progression, demonstrating that CDK1 has a non-canonical role in regulating integrin adhesion complexes and in the migration of cancer cells in 3D interstitial matrix. Here we show that the CDK1 binding partners cyclinB1 and cyclinA2 also have roles in cell migration and invasion in both cancer and non-transformed cells. CyclinB1 plays a key role in RhoA activation to promote rear retraction in a membrane tension dependent manner, while cyclinA2 has a general role in promoting motility. Knockdown of either cyclin significantly perturbs migration with contrasting phenotypes, while knockdown of both together has an additive effect which arrests both migration and division. We find that the cell migration specific role of CDK1 is independent of cell-cycle phase, with inhibition or knockdown of CDK1 perturbing migration in G0/G1 arrested cells, while CDK1-cyclin expression correlates strongly with invasive potential of bladder cancer cell lines. Our findings therefore describe how cyclin-CDK1 complexes orchestrate migration as well as division of cells and that cyclinA2-CDK1 and cyclinB1-CDK1 complexes play distinct roles in motility. Furthermore, these findings suggest that targeting CDK1 signalling in aggressive and invasive tumours may have an unexpected dual potential to combat metastasis in addition to proliferation.

cell biology↗

bioRxiv2022

Peptide Avidity for TCR on CD4 Effectors Determines the Extent of Memory Generation

The impact of initial peptide antigen affinity for TCR in driving memory fate has been studied previously, however its contributions when effectors contract to memory is unclear. To become memory, effector CD4 T cells must recognize antigen at 5-8 days post-infection, at what we call the "effector checkpoint." We examined whether peptide affinity for the TCR of effectors impacts the extent of memory and degree of protection against rechallenge. We made an influenza A virus (IAV) nucleoprotein (NP)-specific TCR transgenic strain, FluNP, and generated NP-peptide variants that bind FluNP TCR with a broad range of avidity. To vary avidity in vivo, we primed naive donor FluNP in IAV-infected hosts, purified 6d FluNP effectors and co-transferred them with peptide-pulsed APC into uninfected second hosts. Higher affinity peptides yielded higher numbers of FluNP memory cells in the spleen and most dramatically in the lung and dLN, and drove better protection against lethal influenza infection. The major impact of avidity was on memory cell number, not cytokine production, and was already apparent within several days of transfer. We previously showed that autocrine IL-2 production during the effector checkpoint prevented default effector apoptosis and supported memory formation. Here, peptide avidity determined the level of IL-2 produced by effectors. IL-2R expression by APC drove more memory cell formation, suggesting that transpresentation of IL-2 by APC at this checkpoint enhanced CD4 memory generation. Secondary memory generation was also avidity-dependent. We propose this pathway selects CD4 effectors of highest affinity to progress to memory.

immunology↗

bioRxiv2021

Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin 6{beta}4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin {beta}4 using siRNA reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, indicating a requirement for 6{beta}4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827.

cancer biology↗

bioRxiv2021

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1

Talin is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behaviour of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronised phosphorylation of a large number of protein targets. CDK1 activity also maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesiveness that are required for successful completion of mitosis. Using a combination of biochemical, structural and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8 through the use of recombinant fragments. Talin also contains a consensus CDK1 phosphorylation motif centred on S1589; a site that was phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of KANK and weakened the mechanical response of the region, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, therefore provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division in multicellular organisms. SummaryThe direct binding of the master cell cycle regulator, CDK1, to the primary integrin effector, talin, provides a primordial solution for coupling the cell proliferation and cell adhesion machineries, and thereby enables microenvironmental control of cell division.

cell biology↗

bioRxiv2020

CD4 Effectors Need to Recognize Antigen Locally to Become Cytotoxic CD4 and Follicular Helper T Cells

Cytotoxic CD4 T cells (ThCTL) are tissue-resident effectors that enhance viral clearance by MHC-II-restricted cytotoxicity of infected cells. Using a model of influenza A virus (IAV) infection, we identify key factors that drive CD4 effectors to differentiate into lung-resident ThCTL. We find that, to become ThCTL, CD4 effectors must again recognize cognate antigen on antigen presenting cells (APC) within the lung. Different APC subsets can drive this transition, including dendritic cells, B cells, and to a lesser extent non-hematopoietic MHC-II+ APC. CD28 co-stimulation is not required and can reduce ThCTL development. In contrast, T follicular helper cells (TFH) that are another specialized CD4 effector subset, require CD28 during this time. Optimal ThCTL generation also requires ongoing infection in the effector phase, that acts independently of antigen presentation. The mechanism involves production of Type I IFN, that induces IL-15 which acts to support further differentiation of CD4 effectors to ThCTL. The multiple spatial, temporal and cellular requirements for ThCTL generation from CD4 effectors described here would be expected to prevent cytotoxic CD4 responses in the lung after pathogen has already been cleared, while ensuring the development of potent lung-restricted ThCTL effectors when pathogen persists.

immunology↗

bioRxiv2020

Adolescent intermittent ethanol exposure induces sex-dependent divergent changes in ethanol drinking and motor activity in adulthood in C57BL/6J mice

With alcohol readily accessible to adolescents, its consumption leads to many adverse effects, including impaired learning, attention, and behavior. Adolescents report higher rates of binge drinking compared to adults. Adolescents are also more prone to substance use disorder during adulthood due to physiological changes during the adolescent developmental period. We used C57BL/6J male and female mice to investigate the long-lasting impact of binge ethanol exposure during adolescence on voluntary ethanol intake and open field behavior during later adolescence and in young adulthood. The present set of experiments were divided into four stages: (1) chronic intermittent vapor inhalation exposure, (2) abstinence, (3) voluntary ethanol intake, and (4) open field behavioral testing. During adolescence, male and female mice were exposed to air or ethanol using an intermittent vapor inhalation with repeated binge pattern ethanol exposure from postnatal day (PND) 28-42. Following this, mice underwent abstinence during late adolescence from PND 43-49 (Experiment 1) or PND 43-69 (Experiment 2). Beginning on PND 49-76 (Experiment 1) or PND 70-97 (Experiment 2), mice were assessed for intermittent voluntary ethanol consumption using a two-bottle drinking procedure over 28 days. Male mice that were exposed to ethanol during adolescence showed increased ethanol consumption during later adolescence (Experiment 1) and in emerging adulthood (Experiment 2), while the female mice showed decreased ethanol consumption. These data demonstrate a sexually divergent shift in ethanol consumption following binge ethanol exposure during adolescence and differences in open field behavior. These data highlight sex-dependent vulnerability to developing substance use disorders in adulthood. Significance StatementCurrently, it is vital to determine the sex-dependent impact of binge alcohol exposure during adolescence, given that until recently females have largely been ignored. Here we show that adolescent male mice that are exposed to binge ethanol during adolescence show long-term changes in behavior in adulthood. In contrast, female mice show a transient decrease in ethanol consumption in adulthood and decreased motor activity spent in the center zone of the open field test. Male mice appear to be more susceptible to the long-term changes in ethanol consumption following binge ethanol exposure during adolescence.

neuroscience↗

bioRxiv2020

Definition of the fibroblast adhesome using multiplexed proximity biotinylation

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry and topological organisation of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.

cell biology↗

bioRxiv2017

Reticular adhesions: A new class of adhesion complex that mediates cell-matrix attachment during mitosis

Adhesion to the extracellular matrix (ECM) persists during mitosis in most cell types. Yet, classical adhesion complexes (ACs), such as focal adhesions and focal complexes, do and must disassemble to enable cytoskeletal rearrangements associated with mitotic rounding. Given this paradox, mechanisms of mitotic cell-ECM adhesion remain undefined. Here, we identify reticular adhesions, a new class of AC that is mediated by integrin v{beta}5, formed during interphase and preserved at cell-ECM attachment sites throughout cell division. Consistent with this role, integrin {beta}5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Quantitative imaging reveals reticular adhesions to be both morphologically and dynamically distinct from classic focal adhesions, while mass spectrometry defines their unique composition; lacking virtually all consensus adhesome components. Indeed, remarkably, reticular adhesions are functionally independent of both talin and F-actin, yet are promoted by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). Overall, the distinct characteristics of reticular adhesions provide a unique solution to the problem of maintaining cell-ECM attachment during mitotic rounding and division.

cell biology↗