Biology subjects

Mitchell, L.

Publications and source records attributed to Mitchell, L..

34 records · Page 2Linked to original sources

bioRxiv2023

Paracrine secretion of MUFAs prevents ferroptosis in triple-negative breast cancer and reveals selenocysteine synthesis dependency for lung metastasis

The limited availability of therapeutic options for patients with triple-negative breast cancer (TNBC) contributes to the high rate of metastatic recurrence and poor prognosis. Ferroptosis is a type of cell death caused by iron-dependent lipid peroxidation and counteracted by the antioxidant activity of the selenoprotein GPX4. Here, we show that TNBC cells secrete an anti-ferroptotic factor in the extracellular environment when cultured at high cell densities but are primed to ferroptosis when forming colonies at low density. We found that secretion of the anti-ferroptotic factors, identified as monounsaturated fatty acid (MUFA) containing lipids, and the vulnerability to ferroptosis of single cells depends on the low expression of stearyl-CoA desaturase (SCD) that is proportional to cell density. Finally, we show that the inhibition of Sec-tRNAsec biosynthesis, an essential step for selenoprotein production, causes ferroptosis and impairs the lung seeding of circulating TNBC cells that are no longer protected by the MUFA-rich environment of the primary tumour.

cancer biology↗

bioRxiv2023

Optimisation of sample preparation from primary mouse tissue to maintain RNA integrity for methods examining translational control

Protein output of different mRNAs can vary by two orders of magnitude therefore it is critical to understand the processes that control gene expression that operate at the level of translation. Translatome-wide techniques such as polysome profiling and ribosome profiling are key methods for determining translations rates occurring on specific mRNAs. These techniques are now widely used in cell lines; however, they are underutilised in tissues and cancer models. Ribonuclease (RNase) expression is often found to be higher in complex primary tissues in comparison to cell lines. Methods used to preserve RNA during lysis often use denaturing conditions which needs to be avoided when requiring to maintain the interaction and position of the ribosome with the mRNA. Here we detail the cell lysis conditions that produce high quality RNA from several different tissues covering a range of endogenous RNase expression levels. We highlight the importance of RNA integrity for accurate determination of the global translation status of the cell as determined by polysome gradients and discuss key aspects to optimise for accurate assessment of the translatome from primary mouse tissue.

molecular biology↗

bioRxiv2023

Biochemical characterization of mRNA capping enzyme from Faustovirus

The mammalian mRNA 5 cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5- mediated mRNA turnover. Hence, installation of the proper 5 cap is crucial in therapeutic applications of synthetic mRNA. The core 5 cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5 triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity towards RNA containing secondary structures, and broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

biochemistry↗

bioRxiv2023

Spatial autocorrelation dimension as a potential determinant for the temporal persistence of human atrial and ventricular fibrillation.

Background: Despite being central to atrial fibrillation (AF) and ventricular fibrillation (VF) mechanisms and therapy, the factors governing AF and VF termination are poorly understood. It has been noted that ratio of system size (L) and the two-point spatial correlation length ({xi}2) are associated with time until termination in transient spatiotemporally chaotic systems, but the relationship between these characteristics and termination has not been systematically studied in human AF and VF. Objective: We aimed assess whether the time to cardiac fibrillation termination can be predicted using a novel estimator, the spatial autocorrelation dimension (Di), defined as the ratio of L and {xi}2, in human AF and VF. Methods: Di was computed and compared in a multi-centre, multi-system study with data for sustained versus spontaneously terminating human AF/VF. VF data was collected during coronary-bypass surgery; and AF data during clinically indicated AF ablation. We analyzed: i) VF mapped using a 256-electrode epicardial sock (n=12pts); ii) AF mapped using a 64-electrode constellation basket-catheter (n=15pts); iii) AF mapped using a 16-electrode HD-grid catheter (n=42pts). To investigate temporal fibrillation persistence, the response of AF-episodes to flecainide (n=7pts) was also studied. Results: Spontaneously terminating fibrillation demonstrated a lower Di (P<0.001 all systems). Lower Di was also seen in paroxysmal compared to persistent AF (P=0.002). Post-flecainide, Di decreased over time (P<0.001). Lower Di was also associated with longer-lasting episodes of AF/VF (R2>0.90, P<0.05 in all cases). Using k-means clustering, two distinct clusters and their centroids were identified i) a cluster of spontaneously terminating episodes, and ii) a cluster of sustained epochs. Conclusion: Di predicts the temporal persistence of cardiac fibrillation. This finding provides potentially important insights into a possible common pathway to termination and therapeutic approaches.

physiology↗

bioRxiv2023

Bacterial suppressor-of-copper-sensitivity (Scs) proteins exhibit diverse thiol-disulfide oxidoreductase cellular functions

Disulfide bond (Dsb) proteins catalyse oxidative protein folding governing bacterial survival and virulence. Dsb systems in Escherichia coli K-12 are well-studied, yet what determines dithiol oxidase or disulfide reductase activity remains unknown. Past studies suggest oligomerisation of periplasmic thiol oxidoreductases dictates the direction of thiol catalytic activity. Here, we studied three suppressor-of-copper-sensitivity C (ScsC) Dsb-like proteins known to exist in the reduced state and bind to copper. These proteins adopt different quaternary structures: Salmonella enterica ScsC (StScsC) is monomeric, while ScsC from Proteus mirabilis (PmScsC) and Caulobacter crescentus (CcScsC) are trimeric. When expressed in the model organism E. coli K-12, we showed that all three ScsC proteins exhibit both dithiol oxidation and disulfide reduction activity, despite structural differences. Interestingly, while ScsC reductase function was supported by the canonical E. coli DsbD reductase, oxidase activity depended on environmental oxidation. However, an engineered monomeric PmScsC synergises with E. coli DsbB to gain dithiol oxidase activity at the expense of reductase function. Thus, oligomerisation could be one mechanism by which ScsC proteins avoid interactions with the periplasmic thiol oxidase pathway. This tightly controls their re-oxidation and maintains ScsC proteins in the reduced state required for binding and sequestering toxic levels of cellular copper.

microbiology↗

bioRxiv2022

MenDEL: PCR Primer Design as Constrained Optimization Process

MotivationThe synthesis of large DNA assemblies has applications in biotechnology, and can help us better understand genome biology. These large DNA assemblies are often constructed from many smaller DNA segments, and it is critical to assess that they are correctly assembled. One low cost and rapid method to ensure that the connection between each segment is correct is to use PCR with primer pairs that span assembly junctions. However, the design of PCR primers for large assemblies consisting of multiple segments, and therefore containing multiple assembly junctions, is a challenging process. Rule-based automation of the process often results in finding primers that satisfy general criteria, but are not necessarily the best fit for every particular junction. ResultsWe have developed MenDEL - a web-based DNA design application, that provides a primer pair computation tool for multiple assembly junctions in such a way that for each junction we automatically pick the optimal pair of primers based on user specified constraints. Availability and ImplementationThe MenDEL application is available at https://mendel-isg.nyumc.org to registered users, and the code base for computing junction primers is available at https://github.com/MendelProject/PrimerOptimization.

bioinformatics↗

bioRxiv2022

Super-enhancers require a combination of classical enhancers and novel facilitator elements to drive high levels of gene expression

Super-enhancers (SEs) are a class of compound regulatory elements which control expression of key cell-identity genes. It remains unclear whether they are simply clusters of independent classical enhancers or whether SEs manifest emergent properties and should therefore be considered as a distinct class of element. Here, using synthetic biology and genome editing, we engineered the well characterised erythroid -globin SE at the endogenous -globin locus, removing all SE constituent elements in a mouse embryonic stem cell-line, to create a "blank canvas". This has allowed us to re-build the SE through individual and combinatorial reinsertion of its five elements (R1, R2, R3, Rm, R4), to test the importance of each constituents sequence and position within the locus. Each re-inserted element independently creates a region of open chromatin and binds its normal repertoire of transcription factors; however, we found a high degree of functional interdependence between the five constituents. Surprisingly, the two strongest -globin enhancers (R1 and R2) act sub-optimally both on their own and in combination, and although the other three elements (R3, Rm and R4) exhibit no discernible enhancer activity, they each exert a major positive effect in facilitating the activity of the classical enhancers (R1 and R2). This effect depends not simply on the sequence of each element but on their positions within the cluster. We propose that these "facilitators" are a novel form of regulatory element, important for ensuring the full activity of SEs, but distinct from conventional enhancer elements.

genetics↗

bioRxiv2022

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid and accurate protein production. The inclusion of chemically modified bases into mRNAs has the potential to alter the strength and pattern of hydrogen bonding between mRNAs and aminoacyl-tRNAs to alter protein synthesis. We investigated how the Nl-methylpseudouridine (m1{Psi}) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the ability of codons to react with cognate and near-cognate tRNAs and release factors. We find that the presence of a single m1{Psi} does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1{Psi} can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1{Psi}, and the related naturally occurring pseudouridine ({Psi}) modification, exhibit the ability to both increase and decrease the extent of amino acid misincorporation in a codon-position and tRNA dependent manner. To ascertain the chemical logic for our biochemical and cellular observations, we computationally modeled tRNAIle(GAU) bound to unmodified and m1{Psi}- or {Psi}-modified phenylalanine codons (UUU). Our modeling suggests that changes in the energetics of mRNA:tRNA interactions largely correlate with the context specificity of Ile-miscoding events we observe on {Psi} and m1{Psi} containing Phe codons. These studies reveal that the sequence context of a given modification within an mRNA plays a large role in determining how (and if) the modification impacts the number and distribution of proteoforms synthesized by the ribosome.

biochemistry↗

bioRxiv2022

Glioblastoma exosomes influence glial cell hyaluronic acid deposition to promote invasiveness

BackgroundInfiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo- and radio-therapy. A deeper understanding of the mechanisms invoked by GMB to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. MethodsCRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterised EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived extracellular matrix (ECM) to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. ResultsGBM cells expressing a p53 mutant (p53273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. ConclusionsThis work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue. KEY POINTSThe p53R273H oncogene encourages GBM cells to release EVs containing podocalyxin. Podocalyxin-containing EVs from GBM increase hyaluronic acid production by astrocytes. Hyaluronic acid production by astrocytes drives GBM migration. IMPORTANCE OF THE STUDYThe infiltrative behaviour of glioblastoma (GBM) leads to widespread dissemination of cancer cells throughout the brain. Thus, even following successful resection of the primary tumour these disseminated cells inevitably contribute to post-surgical relapse. In this study, we have discovered a new mechanism through which GBM can release small extracellular vesicles (EVs) to reprogramme extracellular matrix (ECM) production by astrocytes in a way that supports increased invasive behaviour of the GBM cells. Moreover, we have discovered several key components of the pathway which contribute to this EV-mediated GBM-glial cell communication. Principal amongst these, we show that a particular mutant of the p53 tumour suppressor, p53273H drives the release of EVs which foster the deposition of pro-invasive ECM by astrocytes. This study provides mechanistic insight into why brain tumours expressing p53273H are associated with particularly poor patient survival and highlights the possibility of deploying agents which target astrocyte ECM deposition to reduce the morbidity of p53273H- expressing GBM.

cancer biology↗

bioRxiv2021

Cerebellar GABA change during visuomotor adaptation relates to adaptation performance and cerebellar network connectivity: A Magnetic Resonance Spectroscopic Imaging study.

Motor adaptation is crucial for performing accurate movements in a changing environment and relies on the cerebellum. Although cerebellar involvement has been well characterized, the neurochemical changes in the cerebellum underpinning human motor adaptation remain unknown. We used a novel Magnetic Resonance Spectroscopic Imaging (MRSI) technique to measure changes in the inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA) in the human cerebellum during visuomotor adaptation. Participants used their right hand to adapt to a rotated cursor in the scanner, compared with a control task requiring no adaptation. We spatially resolved adaptation-driven GABA changes at the cerebellar nuclei and cerebellar cortex in the left and the right cerebellar hemisphere independently and found that simple right-hand movements increase GABA in the right cerebellar nuclei and decreases GABA in the left. When isolating adaptation-driven GABA changes, we found an increase in GABA in the left cerebellar nuclei and decrease in GABA in the right cerebellar nuclei. Early adaptation-driven GABA change in the right cerebellar nuclei correlated with adaptation performance: Participants showing greater GABA decrease adapted better, suggesting early GABA change is behaviourally relevant. Early GABA change also correlated with functional connectivity change in a cerebellar network: Participants showing greater decreases in GABA showed greater strength increases in cerebellar network connectivity. Results were specific to GABA, to adaptation and to the cerebellar network. This study provides first evidence for plastic changes in cerebellar neurochemistry during motor adaptation. Characterising these naturally occurring neurochemical changes may provide a basis for developing therapeutic interventions to facilitate human motor adaptation. Significance StatementDespite motor adaptation being fundamental to maintaining accurate movements, its neurochemical basis remains poorly understood, perhaps because measuring neurochemicals in the human cerebellum is technically challenging. Using a novel magnetic resonance spectroscopic imaging method, this study provides evidence for GABA changes in the left and right cerebellar nuclei driven by both simple movement and motor adaptation. The adaptation-driven GABA change in the right cerebellar nuclei correlated with adaptation performance, and with functional connectivity change in a cerebellar network. These results show the first evidence for plastic changes in cerebellar neurochemistry during a cerebellar learning task. This provides the basis for developing therapeutic interventions that facilitate these naturally occurring changes to amplify cerebellar-dependent learning.

neuroscience↗

bioRxiv2021

Shorter constant work rate cycling tests as proxies for longer tests in highly trained cyclists

Severe-intensity constant work rate (CWR) cycling tests are useful for monitoring training progression and adaptation as they impose significant physiological and psychological strain and thus simulate the high-intensity competition environment. However, fatiguing tests require substantial recovery and may disrupt athlete training or competition preparation. Therefore, the development of a brief, minimally fatiguing test providing comparable information is desirable. PurposeTo determine whether physiological variables measured during, and functional decline in maximal power output immediately after, a 2-min CWR test can act as a proxy for 4-min test outcomes. MethodsPhysiological stress was monitored and pre-to-post-CWR changes in 10-s sprint power computed (to estimate performance fatigability) during 2- and 4-min CWR tests in high-level cyclists. ResultsThe 2-min CWR test evoked a smaller decline in sprint mechanical power (32% vs. 47%, p<0.001), however both the physiological variables and sprint mechanical power were independently and strongly correlated between 2- and 4-min tests. Differences in V{middle dot}O2peak and blood lactate concentration in both CWR tests were strongly associated with the decline in sprint mechanical power. ConclusionPhysiological variables measured during, and the loss in sprint mechanical power measured after, a severe-intensity 2-min CWR test were less than in the 4-min test. Yet strong correlations between 2- and 4-min test outcomes indicated that the 2-min test can be used as a proxy for the longer test. Because shorter tests are less strenuous, they should have less impact on training and competition preparation and may therefore be more practically applicable within the elite performance environment.

physiology↗

bioRxiv2021

TRAK proteins encode distinct MIRO-dependent and MIRO-independent mechanisms for associating with the mitochondrial outer membrane

Current models suggest that MIRO GTPases anchor cytoskeletal motors to the mitochondrial outer membrane (MOM). However, our previous findings indicate that the unconventional myosin, MYO19, interacts with MIRO weakly and that a MIRO-independent MOM-localizing domain interacts more tightly with the MOM. To test the hypothesis that other MIRO interactors may also have MIRO-independent MOM-binding, we examined interactions between TRAK proteins (microtubule motor-mitochondria adaptor proteins) and the MOM via quantitative fluorescence microscopy and steady-state kinetic approaches. Using GFP-TRAK truncations expressed in MIRO1-2 double knockout mouse embryonic fibroblasts, we identified a MIRO-independent mitochondrial binding domain in the C-terminus of TRAK1 and TRAK2, sufficient for MOM-localization similar to what we observed for full length GFP-TRAK proteins. The MIRO-binding domains (MBD) of the TRAK proteins were only able to localize to mitochondria in the presence of ectopic expression of MIRO. Importantly, fluorescence recovery after photobleaching (FRAP) demonstrated that the steady-state kinetics of TRAKMBD/MIRO interactions were faster-exchanging than for either full-length TRAK or the TRAK C-terminal MOM-binding domain expressed alone. These data support a model where faster-exchanging TRAK/MIRO associations could support initial association and/or TRAK activation, while MIRO-independent binding contributes significantly to tighter association to the MOM.

cell biology↗

bioRxiv2021

NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

The new next-generation sequencing platforms by Oxford Nanopore Technologies for direct RNA sequencing (direct RNA-seq) allow for an in-depth and comprehensive study of the epitranscriptome by enabling direct base calling of RNA modifications. Non-coding RNAs constitute the most frequently documented targets for RNA modifications. However, the current standard direct RNA-seq approach is unable to detect many of these RNAs. Here we present NERD-seq, a sequencing approach which enables the detection of multiple classes of non-coding RNAs excluded by the current standard approach. Using total RNA from a tissue with high known transcriptional and non-coding RNA activity in mouse, the brain hippocampus, we show that, in addition to detecting polyadenylated coding and non-coding transcripts as the standard approach does, NERD-seq is able to significantly expand the representation for other classes of RNAs such as snoRNAs, snRNAs, scRNAs, srpRNAs, tRNAs, rRFs and non-coding RNAs originating from LINE L1 elements. Thus, NERD-seq presents a new comprehensive direct RNA-seq approach for the study of epitranscriptomes in brain tissues and beyond.

genomics↗

bioRxiv2020

Quantitative in vivo bioluminescence imaging of orthotopic patient-derived glioblastoma xenografts

Despite extensive research, little progress has been made in glioblastoma therapy, owing in part to a lack of adequate preclinical in vivo models to study this disease. To mitigate this, primary patient-derived cell lines, which maintain their specific stem-like phenotypes, have replaced established glioblastoma cell lines. However, due to heterogenous tumour growth inherent in glioblastoma, the use of primary cells for orthotopic in vivo studies often requires large experimental group sizes. Therefore, when using intracranial patient-derived xenograft (PDX) approaches, it is advantageous to deploy imaging techniques to monitor tumour growth and allow stratification of mice. Here we show that stable expression of near-infrared fluorescent protein (iRFP) in patient-derived glioblastoma cells enables rapid direct non-invasive monitoring of tumour development without compromising tumour stemness or tumorigenicity. Moreover, as this approach does not depend on the use of agents like luciferin, which can cause variability due to changing bioavailability, it can be used for quantitative longitudinal monitoring of tumour growth. Notably, we show that this technique also allows quantitative assessment of tumour burden in highly invasive models spreading throughout the brain. Thus, iRFP transduction of primary patient-derived glioblastoma cells is a reliable, cost- and time-effective way to monitor heterogenous orthotopic PDX growth.

cancer biology↗

bioRxiv2019

Renewal theory provides a universal quantitative framework to characterise the continuous regeneration of rotational events in cardiac fibrillation

BackgroundCardiac fibrillation is thought to be maintained by rotational activity, with pivoting regions called phase singularities (PSs). Despite a century of research, no clear quantitative framework exists to model the fundamental processes responsible for the continuous formation and destruction of rotors in fibrillation.\n\nObjectiveWe conducted a multi-modality, multi-species study of AF/VF under the hypothesis that PS formation/destruction in fibrillation can be modelled as self-regenerating renewal processes, producing exponential distributions of inter-event times governed by constant rate-parameters defined by the prevailing properties of the system.\n\nMethodsPS formation/destruction was studied and cross-validated in 5 models, using basket recordings and optical mapping from: i) human persistent AF (n = 20), ii) tachypaced sheep AF (n = 5), iii) rat AF (n = 4), iv) rat VF (n = 11) and v) computer simulated AF (SIM). Hilbert phase maps were constructed. PS lifetime data were fitted by exponential probability distribution functions (PDFs) computed using maximum entropy theory, and the rate parameter ({lambda}) determined. A systematic review was conducted to cross-validate with source data from literature.\n\nResultsPS destruction/formation distributions showed good fits to an exponential in all systems (R2 [≥] 0.90). In humans,{lambda} = 4.6%/ms (95%CI,4.3,4.9)), sheep 4.4%/ms (95%CI,4.1,4.7)), rat AF 38%/ms (95%CI,22,55), rat VF 46%/ms (95%CI,31.2,60.2) and SIM 5.4%/ms (95%CI,4.1,6.7). All PS distributions identified through systematic review were exponential with {lambda} comparable to experimental data.\n\nConclusionThese results provide a universal quantitative framework to explain rotor formation and destruction in AF/VF, and a platform for therapeutic advances in cardiac fibrillation.

physiology↗

bioRxiv2018

Connecting surveillance and population-level influenza incidence

There is substantial interest in estimating and forecasting influenza incidence. Surveillance of influenza is challenging as one needs to demarcate influenza from other respiratory viruses, and due to asymptomatic infections. To circumvent these challenges, surveillance data often targets influenza-like-illness, or uses context-specific normalisations such as test positivity or per-consultation rates. Specifically, influenza incidence itself is not reported. We propose a framework to estimate population-level influenza incidence, and its associated uncertainty, using surveillance data and hierarchical observation processes. This new framework, and forecasting and forecast assessment methods, are demonstrated for three Australian states over 2016 and 2017. The framework allows for comparison within and between seasons in which surveillance effort has varied. Implementing this framework would improve influenza surveillance and forecasting globally, and could be applied to other diseases for which surveillance is difficult.

epidemiology↗