Biology subjects

Graham, S.

Publications and source records attributed to Graham, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Antiviral Type III CRISPR signalling via conjugation of ATP and AdoMet

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements (MGE) 1, 2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA (crRNA) to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against MGE either directly, via the integral HD nuclease domain 3-5 or indirectly, via synthesis of cyclic oligonucleotide (cOA) second messengers to activate diverse ancillary effectors 6-9. A subset of type III CRISPR systems encode an uncharacterised CorA-family membrane protein and an associated NrN family phosphodiesterase predicted to function in antiviral defence. Here, we demonstrate that the CorA associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against MGE when expressed inE. coli. However, B. fragilis Cmr does not synthesise cOA species on activation, instead generating a previously undescribed sigalling molecule, SAM-AMP (3-adenylyl-AdoMet) by conjugating ATP to S-adenosyl methionine via a phosphodiester bond. Once synthesised, SAM-AMP binds to the CorA effector, presumably leading to cell death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR associated phosphodiesterases or a SAM-AMP lyase, providing an "off switch" analogous to cOA specific ring nucleases 10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

microbiology↗

bioRxiv2023

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

cancer biology↗

bioRxiv2022

Structure of the Saccharolobus solfataricus type III-D CRISPR effector

CRISPR-Cas is a prokaryotic adaptive immune system, classified into six different types, each characterised by a signature protein. Type III systems, classified based on the presence of a Cas10 subunit, are rather diverse multi-subunit assemblies with a range of enzymatic activities and downstream ancillary effectors. The broad array of current biotechnological CRISPR applications is mainly based on proteins classified as Type II, however recent developments established the feasibility and efficacy of multi-protein Type III CRISPR-Cas effector complexes as RNA-targeting tools in eukaryotes. The crenarchaeon Saccharolobus solfataricus has two type III system subtypes (III-B and III-D). Here, we report the cryo-EM structure of the Csm Type III-D complex from S. solfataricus (SsoCsm), which uses CRISPR RNA to bind target RNA molecules, activating the Cas10 subunit for antiviral defence. The structure reveals the complex organisation, subunit/subunit connectivity and protein/guide RNA interactions of the SsoCsm complex, one of the largest CRISPR effectors known.

microbiology↗

bioRxiv2022

Sensitivity of diffusion-tensor and correlated diffusion imaging to white-matter microstructural abnormalities: application in COVID-19

There has been growing attention on the effect of COVID-19 on white-matter microstructure, especially among those that self-isolated after being infected. There is also immense scientific interest and potential clinical utility to evaluate the sensitivity of single-shell diffusion MRI methods for detecting such effects. In this work, the sensitivities of three single-shell-compatible diffusion MRI modeling methods are compared for detecting the effect of COVID-19, including diffusion-tensor imaging, diffusion-tensor decomposition of orthogonal moments and correlated diffusion imaging. Imaging was performed on self-isolated patients at baseline and 3-month follow-up, along with age- and sex-matched controls. We demonstrate through simulations and experimental data that correlated diffusion imaging is associated with far greater sensitivity, being the only one of the three single-shell methods to demonstrate COVID-19-related brain effects. Results suggest less restricted diffusion in the frontal lobe in COVID-19 patients, but also more restricted diffusion in the cerebellar white matter, in agreement with several existing studies highlighting the vulnerability of the cerebellum to COVID-19 infection. These results, taken together with the simulation results, suggest that a significant proportion of COVID-19 related white-matter microstructural pathology manifests as a change in water diffusivity. Interestingly, different b-values also confer different sensitivities to the effects. No significant difference was observed in patients at the 3-month follow-up, likely due to the limited size of the follow-up cohort. To summarize, correlated diffusion imaging is shown to be a sensitive single-shell diffusion analysis approach that allows us to uncover opposing patterns of diffusion changes in the frontal and cerebellar regions of COVID-19 patients, suggesting the two regions react differently to viral infection.

biophysics↗

bioRxiv2022

The immune response to lumpy skin disease virus in cattle is influenced by inoculation route

Lumpy skin disease virus (LSDV) causes severe disease in cattle and water buffalo and is transmitted by hematophagous arthropod vectors. Detailed information of the adaptive and innate immune response to LSDV is limited, hampering the development of tools to control the disease. This study provides an in-depth analysis of the immune responses of calves experimentally inoculated with LSDV via either needle-inoculation or arthropod-inoculation using virus-positive Stomoxys calcitrans and Aedes aegypti vectors. Seven out of seventeen needle-inoculated calves (41%) developed clinical disease characterised by multifocal necrotic cutaneous nodules. In comparison 8/10 (80%) of the arthropod-inoculated calves developed clinical disease. A variable LSDV-specific IFN-{gamma} immune response was detected in the needle-inoculated calves from 5 days post inoculation (dpi) onwards, with no difference between clinical calves (developed cutaneous lesions) and nonclinical calves (did not develop cutaneous lesions). In contrast a robust and uniform cell-mediated immune response was detected in all eight clinical arthropod-inoculated calves, with little response detected in the two nonclinical arthropod-inoculated calves. Neutralising antibodies against LSDV were detected in all inoculated cattle from 5-7 dpi. Comparison of the production of anti-LSDV IgM and IgG antibodies revealed no difference between clinical and nonclinical needle-inoculated calves, however a strong IgM response was evident in the nonclinical arthropod-inoculated calves but absent in the clinical arthropod-inoculated calves. This suggests that early IgM production is a correlate of protection in LSD. This study presents the first evidence of differences in the immune response between clinical and nonclinical cattle and highlights the importance of using a relevant transmission model when studying LSD.

immunology↗

bioRxiv2022

Structure and Neutralization Mechanism of a Human Antibody Targeting a Complex Epitope on Zika Virus

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

microbiology↗

bioRxiv2022

Structure and mechanism of the type I-G CRISPR effector

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3 end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. Type I-G Cascade is one of the most streamlined Class 1 CRISPR effectors. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

microbiology↗

bioRxiv2022

Crowding induced morphological changes in synthetic lipid vesicles determined using smFRET

Lipid vesicles are valuable mesoscale molecular confinement vessels for studying membrane mechanics and lipid-protein interactions, and they have found utility among bio-inspired technologies including drug delivery vehicles. While vesicle morphology can be modified by changing the lipid composition and introducing fusion or pore-forming proteins and detergents, the influence of extramembrane crowding on vesicle morphology has remained under explored owing to a lack of experimental tools capable of capturing morphological changes on the nanoscale. Here, we use biocompatible polymers to simulate molecular crowding in vitro, and through combinations of FRET spectroscopy, lifetime analysis, dynamic light scattering and single-vesicle imaging, we characterize how crowding regulates vesicle morphology. We show that both freely-diffusing and surface-tethered vesicles fluorescently tagged with the DiI and DiD FRET pair undergo compaction in response to modest concentrations of sorbitol, polyethylene glycol and Ficoll. A striking observation is that sorbitol results in irreversible compaction, whereas the influence of high molecular weight PEG-based crowders was found to be reversible. Regulation of molecular crowding allows for precise control of vesicle architecture in vitro, with vast implications for drug delivery and vesicle trafficking systems. Furthermore, our observations of vesicle compaction may also serve to act as a mechanosensitive readout of extramembrane crowding.

biophysics↗

bioRxiv2022

Cyclic nucleotide-induced superhelical structure activates a bacterial TIR immune effector

Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1, which originated in bacteria 2. These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3. One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD+ when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6. Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA3) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD+ degradation. Our study illuminates a striking example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

biochemistry↗

bioRxiv2021

TIAToolbox: An End-to-End Toolbox for Advanced Tissue Image Analytics

Computational Pathology (CPath) has seen rapid growth in recent years, driven by advanced deep learning (DL) algorithms. These algorithms typically share the same sequence of steps. However, due to the sheer size and complexity of handling large multi-gigapixel whole-slide images, there is no open-source software library that provides a generic end-to-end API for pathology image analysis using best practices for CPath. Most researchers have designed custom pipelines from the bottom-up, restricting the development of advanced CPath algorithms to specialist users. To help overcome this bottleneck, we present TIAToolbox, a Python toolbox designed to make CPath more accessible to new and advanced CPath scientists and pathologists alike. We provide a usable and adaptable library with efficient, cutting-edge and unit-tested tools for data loading, pre-processing, model inference, post-processing and visualization. This enables all kinds of users to easily build upon recent DL developments in the CPath literature. TIAToolbox provides a user-friendly modular API to enable seamless integration of advanced DL algorithms. We show with the help of examples how state-of-the-art DL algorithms can be streamlined using TIAToolbox.

bioengineering↗

bioRxiv2020

A novel antigenic site spanning domains I and III of the Zika virus envelope glycoprotein is the target of strongly neutralizing human monoclonal antibodies

Zika virus (ZIKV), a mosquito-transmitted flavivirus, caused a large epidemic in Latin America between 2015 and 2017. Effective ZIKV vaccines and treatments are urgently needed to prevent future epidemics and severe disease sequelae. People infected with ZIKV develop strongly neutralizing antibodies linked to viral clearance and durable protective immunity. To understand mechanisms of protective immunity and to support the development of ZIKV vaccines, here we characterize the properties of a strongly neutralizing antibody, B11F, isolated from a recovered ZIKV patient. Our results indicate that B11F targets a complex epitope on the virus that spans domains I and III of the envelope glycoprotein. While previous studies point to quaternary epitopes centered on domain II of ZIKV E glycoprotein as targets of strongly neutralizing and protective human antibodies, we uncover a new site spanning domain I and III as a target of strongly neutralizing human antibodies. ImportancePeople infected with Zika virus develop durable neutralizing antibodies that prevent repeat infections. In the current study, we characterize a ZIKV-neutralizing human monoclonal antibody isolated from a patient after recovery. Our studies establish a novel site on the viral envelope targeted by human neutralizing antibodies. Our results are relevant to understanding how antibodies block infection and for guiding the design and evaluation of candidate vaccines.

immunology↗

bioRxiv2020

Elevated temperature inhibits SARS-CoV-2 replication in respiratory epithelium independently of the induction of IFN-mediated innate immune defences

The pandemic spread of SARS-CoV-2, the etiological agent of COVID-19, represents a significant and ongoing international health crisis. A key symptom of SARS-CoV-2 infection is the onset of fever, with a hyperthermic temperature range of 38 to 41{degrees}C. Fever is an evolutionarily conserved host response to microbial infection and inflammation that can influence the outcome of viral pathogenicity and regulation of host innate and adaptive immune responses. However, it remains to be determined what effect elevated temperature has on SARS-CoV-2 tropism and replication. Utilizing a 3D air-liquid interface (ALI) model that closely mimics the natural tissue physiology and cellular tropism of SARS-CoV-2 infection in the respiratory airway, we identify tissue temperature to play an important role in the regulation of SARS-CoV-2 infection. We show that temperature elevation induces wide-spread transcriptome changes that impact upon the regulation of multiple pathways, including epigenetic regulation and lncRNA expression, without disruption of general cellular transcription or the induction of interferon (IFN)-mediated antiviral immune defences. Respiratory tissue incubated at temperatures >37{degrees}C remained permissive to SARS-CoV-2 infection but severely restricted the initiation of viral transcription, leading to significantly reduced levels of intraepithelial viral RNA accumulation and apical shedding of infectious virus. To our knowledge, we present the first evidence that febrile temperatures associated with COVID-19 inhibit SARS-CoV-2 replication. Our data identify an important role for temperature elevation in the epithelial restriction of SARS-CoV-2 that occurs independently of the induction of canonical IFN-mediated antiviral immune defences and interferon-stimulated gene (ISG) expression.

microbiology↗

bioRxiv2020

Gut microbiome dysbiosis is associated with elevated toxic bile acids in Parkinson's disease

The gut microbiome can impact brain health and is altered in Parkinsons disease (PD) patients. Here, we investigate changes in the functional microbiome in the appendix of PD patients relative to controls by metatranscriptomic analysis. We find microbial dysbiosis affecting lipid metabolism, particularly an upregulation of bacteria responsible for secondary bile acid synthesis. Proteomic and transcript analysis corroborates a disruption in cholesterol homeostasis and lipid catabolism. Bile acid analysis reveals an increase in microbially-derived, toxic secondary bile acids. Synucleinopathy in mice induces similar microbiome alterations to those of PD patients. The mouse model of synucleinopathy has elevated DCA and LCA. An analysis of blood markers shows evidence of biliary abnormalities early in PD, including elevated alkaline phosphatase and bilirubin. Increased bilirubin levels are also evident before PD diagnosis. In sum, microbially-derived toxic bile acids are heightened in PD and biliary changes may even precede the onset of overt motor symptoms.

neuroscience↗

bioRxiv2020

A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain induces potent neutralising antibody responses

There is dire need for an effective and affordable vaccine against SARS-CoV-2 to tackle the ongoing pandemic. In this study, we describe a modular virus-like particle vaccine candidate displaying the SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) using SpyTag/SpyCatcher technology (RBD-SpyVLP). Low doses of RBD-SpyVLP in a prime-boost regimen induced a strong neutralising antibody response in mice and pigs that was superior to convalescent human sera. We evaluated antibody quality using ACE2 blocking and neutralisation of cell infection by pseudovirus or wild-type SARS-CoV-2. Using competition assays with a monoclonal antibody panel, we showed that RBD-SpyVLP induced a polyclonal antibody response that recognised all key epitopes on the RBD, reducing the likelihood of selecting neutralisation-escape mutants. The induction of potent and polyclonal antibody responses by RBD-SpyVLP provides strong potential to address clinical and logistic challenges of the COVID-19 pandemic. Moreover, RBD-SpyVLP is highly resilient, thermostable and can be lyophilised without losing immunogenicity, to facilitate global distribution and reduce cold-chain dependence.

immunology↗

bioRxiv2020

Experimental glaucoma retinal proteomics identifies mutually exclusive and overlapping molecular characteristics with human glaucoma

Current evidence suggests that exposure to chronically induced intraocular pressure (IOP) leads to neurodegenerative changes in the inner retina. This study aimed to determine retinal proteomic alterations in a rat model of glaucoma and compared findings with human retinal proteomics changes in glaucoma reported previously. We developed an experimental glaucoma rat model by subjecting the rats to increased IOP (9.3{+/-}0.1 vs 20.8{+/-}1.6 mm Hg) by weekly microbead injections into the eye (8 weeks). The retinal tissues were harvested from control and glaucomatous eyes and protein expression changes analysed using multiplexed quantitative proteomics approach. Immunofluorescence was performed for selected protein markers for data validation. Our study identified 4304 proteins in the rat retinas. Out of these, 139 proteins were downregulated ([≤]0.83) while expression of 109 proteins was upregulated ([≥]1.2-fold change) under glaucoma conditions (p[≤]0.05). Computational analysis revealed reduced expression of proteins associated with glutathione metabolism, mitochondrial dysfunction/oxidative phosphorylation, cytoskeleton and actin filament organisation, along with increased expression coagulation cascade, apoptosis, oxidative stress and RNA processing markers. Further functional network analysis highlighted the differential modulation of nuclear receptor signalling, cellular survival, protein synthesis, transport and cellular assembly pathways. Alterations in crystallin family, glutathione metabolism and mitochondrial dysfunction associated proteins shared similarities between the animal model of glaucoma and the human disease condition. In contrast, the activation of the classical complement pathway and upregulation of cholesterol transport proteins, were exclusive to the human glaucoma. These findings provide insights into the neurodegenerative mechanisms that are specifically affected in the retina in response to chronically elevated IOP.

neuroscience↗

bioRxiv2020

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.

biochemistry↗

bioRxiv2020

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. To avoid this fate, some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The activities of the two constituent enzymes in the fusion protein cooperate to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

biochemistry↗

bioRxiv2020

Cerebral Atrophy in Amyotrophic Lateral Sclerosis Parallels the Pathological Distribution of TDP43

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a preferential involvement of both upper and lower motor neurons. Evidence from neuroimaging and post-mortem studies confirms additional involvement of brain regions extending beyond the motor cortex. The aim of this study was to assess the extent of cerebral disease in ALS cross-sectionally and longitudinally, and to compare the findings with a recently proposed disease-staging model of ALS pathology. Deformation-based morphometry (DBM) was used to identify the patterns of brain atrophy associated with ALS and to assess their relationship with clinical symptoms. Longitudinal T1-weighted MRI data and clinical measures were acquired at baseline, 4 months, and 8 months, from 66 ALS patients and 43 age-matched controls who participated in the Canadian ALS Neuroimaging Consortium (CALSNIC) study. Whole brain voxel-wise mixed-effects modelling analysis showed extensive atrophy patterns differentiating ALS patients from the normal controls. Cerebral atrophy was present in the motor cortex and corticospinal tract, involving both GM and WM, and to a lesser extent in non-motor regions. More specifically, the results showed significant bilateral atrophy in the motor cortex, the corticospinal tract including the internal capsule and brainstem, with an overall pattern of ventricular enlargement; along with significant progressive longitudinal atrophy in the precentral gyrus, frontal and parietal white matter, accompanied by ventricular and sulcal enlargement. Atrophy in the precentral gyrus was significantly associated with greater disability as quantified with the ALS Functional Rating Scale-Revised (ALSFRS-R) (p<0.0001). The pattern of atrophy observed using DBM was consistent with the Brettschneiders four stage pathological model of the disease. Deformation based morphometry provides a sensitive indicator of atrophy in ALS, and has potential as a biomarker of disease burden, in both gray and white matter.

neuroscience↗