Biology subjects

Howe, J.

Publications and source records attributed to Howe, J..

10 recordsLinked to original sources

bioRxiv2025

Alignment between Duplex Sequencing and transgenic rodent mutation assay data in the assessment of in vivo NDMA-induced mutagenesis

The nitrosamine N-nitrosodimethylamine (NDMA) is a mutagen and rodent carcinogen that has been identified as a process impurity in some commercially available medicines, leading to market withdrawals and new impurity control measures. Error-corrected DNA sequencing techniques, such as Duplex Sequencing (DS), have error rates low enough to revolutionise genetic toxicology testing by directly measuring in vivo mutagenesis within days of exposure. Here, DS was performed on liver samples from an OECD-compliant, Transgenic Rodent Gene Mutation Assay (TGR) conducted under GLP standards. MutaMouse specimens were orally dosed with NDMA using either a repeat-dose 28-day regimen (0.02-4 mg/kg(bw)/day) or single bolus doses of either 5 or 10 mg/kg(bw) administered on day one. Dose-dependent increases in mutation frequency were detected by DS in liver, enabling a No-Observed Genotoxic Effect Level (NOGEL) of 0.07 mg/kg(bw)/day to be determined, supported by mechanistic analyses of trinucleotide mutation spectra. Benchmark dose (BMD) modelling determined similar BMD50 values from either DS or TGR, demonstrating concordance across the two techniques albeit with greater precision from DS due to smaller inter-animal variation. DS offers a fundamental change in mutagenicity assessments enabling more precise point-of-departure determinations with mechanistic clarity and 3Rs advantages compared to the standard TGR approach.

pharmacology and toxicology↗

bioRxiv2024

LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers

53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.

biophysics↗

bioRxiv2023

Visualization of syntrophic benzene-fermenting Desulfobacterota ORM2 in a methanogenic enrichment culture using fluorescence in situ hybridization

Although benzene degradation under strictly anoxic conditions was first reported over 25 years ago, the mechanism for benzene activation in the absence of oxygen is still elusive. A major limitation has been the difficulty to grow anaerobic benzene-degrading enrichment cultures. Our laboratory has maintained a methanogenic enrichment culture for decades, harboring a benzene fermenter referred to as Desulfobacterota ORM2. Recent genomic analyses indicate that ORM2 is not affiliated with any characterized genus, but it is phylogenetically similar to several other known and predicted benzene degraders. Desulfobacterota ORM2 has a doubling time of approximately 30 days and often enters a long lag or decay phase after inoculation into sterile pre-reduced anaerobic medium. A specific fluorescent in situ hybridization (FISH) probe was used to observe Desulfobacterota ORM2 cells during this decay phase, revealing a rod-shaped cell of variable length with a tendency to associate with other cells, particularly methanogens. Microscopic and genomic analyses indicate that Desulfobacterota ORM2 may produce extracellular polymeric substances (EPS) that likely contribute to cell aggregation. The production of EPS may consume a significant amount of energy, perhaps contributing to the lag time before onset of growth of Desulfobacterota ORM2 post-inoculation. We observed little cell aggregation in a culture amended with very high concentrations of benzene (90-120 mg/L). This study visualized the cells of a novel clade within the Desulfobacterota for the first time, enabling monitoring of spatial organization within a methanogenic consortium and provides hints to improve the growth rate of ORM2. ImportanceA specific FISH probe was designed for the poorly characterized benzene fermenter Desulfobacterota ORM2. This probe was used to monitor changes in spatial organization in a methanogenic benzene-degrading enrichment culture. ORM2 cells were often found in cell aggregates, revealing a possible reason for the long lag phases observed after inoculation.

microbiology↗

bioRxiv2022

Candidatus Nealsonbacteria (OD1) in a methanogenic benzene-degrading enrichment culture is likely an ectosymbiotic biomass recycler

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance of Ca. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate Ca. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed that Ca. Nealsonbacteria DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other Ca. Nealsonbacteria found in anoxic environments.

microbiology↗

bioRxiv2022

Impaired OTUD7A-dependent Ankyrin regulation mediates neuronal dysfunction in mouse and human models of the 15q13.3 microdeletion syndrome

Copy number variations (CNV) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

neuroscience↗

bioRxiv2021

Network diffusion model predicts neurodegeneration in limb-onset amyotrophic lateral sclerosis

The trans-neural propagation of phosphorylated 43-kDa transactive response DNA-binding protein (pTDP-43) contributes to neurodegeneration in Amyotrophic Lateral Sclerosis (ALS). We investigated whether Network Diffusion Model (NDM), a biophysical model of spread of pathology via the brain connectome, could capture the severity and progression of neurodegeneration (atrophy) in ALS. We measured degeneration in limb-onset ALS patients (n=14 at baseline, 12 at 6-months, and 9 at 12 months) and controls (n=12 at baseline) using FreeSurfer analysis on the structural T1-weighted Magnetic Resonance Imaging (MRI) data. The NDM was simulated on the canonical structural connectome from the IIT Human Brain Atlas. To determine whether NDM could predict the atrophy pattern in ALS, the accumulation of pathology modelled by NDM was correlated against atrophy measured using MRI. The cross-sectional analyses revealed that the network diffusion seeded from the inferior frontal gyrus (pars triangularis and pars orbitalis) significantly predicts the atrophy pattern in ALS compared to controls. Whereas, atrophy over time with-in the ALS group was best predicted by seeding the network diffusion process from the inferior temporal gyrus at 6-month and caudal middle frontal gyrus at 12-month. Our findings suggest the involvement of extra-motor regions in seeding the spread of pathology in ALS. Importantly, NDM was able to recapitulate the dynamics of pathological progression in ALS. Understanding the spatial shifts in the seeds of degeneration over time can potentially inform further research in the design of disease modifying therapeutic interventions in ALS.

neuroscience↗

bioRxiv2020

Automated and statistically corrected identification of flexible multivalent IDP-bound assemblies in electron micrographs

Multivalent intrinsically disordered protein (IDP) complexes are prevalent in biology and control diverse cellular functions, including tuning levels of transcription, coordinating cell-signaling events, and regulating the assembly and disassembly of complex macromolecular architectures. These systems pose a significant challenge to structural investigation, due to the continuum dynamics imparted by the IDP and compositional heterogeneity resulting from characteristic low-affinity interactions. Traditional single-particle electron microscopy (EM) is a powerful tool for visualizing IDP complexes. However, the IDPs themselves are typically "invisible" by EM, undermining methods of image analysis and structural interpretation. To overcome these challenges, we developed a pipeline for automated analysis of common beads-on-a-string type of assemblies, composed of IDPs bound at multivalent sites to the ubiquitous ~20 kDa cross-linking hub protein LC8. This approach quantifies conformational and compositional heterogeneity on a single-particle basis, and statistically corrects spurious observations arising from random proximity of bound and unbound LC8. After careful validation of the methodology, the approach was applied to the nuclear pore IDP Nup159 and the transcription factor ASCIZ. The analysis unveiled significant compositional and conformational diversity in both systems that could not be obtained from traditional single particle EM class-averaging strategies, and shed new light on how these architectural properties contribute to their physiological roles in supramolecular assembly and transcriptional regulation. Ultimately, we expect that this approach may be adopted to many other intrinsically disordered systems that have evaded traditional methods of structural characterization. Significance StatementIntrinsically disordered proteins (IDPs) or protein regions (IDRs) represent >30% of the human proteome, but mechanistically remain some of the most poorly understood classes of proteins in biology. This dearth in understanding stems from these very same intrinsic and dynamic properties, which make them difficult targets for quantitative and structural characterization. Here, we present an automated approach for extracting quantitative descriptions of conformational and compositional heterogeneity present in a common beads-on-a-string type of multivalent IDP system from single-particle images in electron micrographs. This promising approach may be adopted to many other intrinsically disordered systems that have evaded traditional ensemble methods of characterization.

biophysics↗

bioRxiv2019

Serial assessment of iron in the motor cortex in limb-onset Amyotrophic Lateral Sclerosis using Quantitative Susceptibility Mapping

ObjectiveDysregulation of iron in the cerebral motor areas has been hypothesized to occur in individuals with Amyotrophic Lateral Sclerosis (ALS). There is still limited knowledge regarding iron dysregulation in the progression of ALS pathology. Our objectives were to use magnetic resonance based Quantitative Susceptibility Mapping (QSM) to investigate the association between iron dysregulation in the motor cortex and clinical manifestations in patients with limb-onset ALS, and to examine changes in the iron concentration in the motor cortex in these patients over a six-month period. MethodsIron concentration was investigated using magnetic resonance based -QSM in the primary motor cortex and the pre-motor area in thirteen limb-onset ALS patients (including five lumbar onset, six cervical onset and two flail arm patients), and eleven age and sex-matched healthy controls. Nine ALS patients underwent follow-up scans at six months. ResultsSignificantly increased QSM was observed in the left posterior primary motor area (p = 0.02, Cohens d = 0.9) and right anterior primary motor area (p = 0.02, Cohens d = 0.92) in all individuals with limb-onset ALS compared to healthy controls. Increased QSM was observed in the primary motor and pre-motor area at baseline in patients with lumbar onset ALS patients, but not cervical limb-onset ALS patients, compared to healthy controls. No significant change in QSM was observed at the six-month follow-up scans in the ALS patients. ConclusionsThe findings suggest that iron dysregulation can be detected in the motor cortex in limb-onset ALS, which does not appreciably change over a further 6 months. Individuals with lumbar onset ALS appear to be more susceptible to motor cortex iron dysregulation compared to the individuals with cervical onset ALS. Importantly, this study highlights the potential use of QSM as a radiological indicator in disease diagnosis, and in clinical trials in limb-onset ALS and its subtypes. HighlightsO_LISerial measurement of QSM in the motor cortex in limb-onset ALS was performed C_LIO_LIQSM changes in the motor cortex in ALS sub-groups were investigated C_LIO_LIHigher QSM was observed in the motor cortex in Lumbar ALS relative to controls C_LIO_LIQSM is sensitive to iron dysregulation in the motor cortex in limb-onset ALS C_LI

neuroscience↗

bioRxiv2019

Control iPSC lines with clinically annotated genetic variants for versatile multi-lineage differentiation

Induced Pluripotent Stem Cells (iPSC) derived from healthy individuals are important controls for disease modeling studies. To create a resource of genetically annotated iPSCs, we reprogrammed footprint-free lines from four volunteers in the Personal Genome Project Canada (PGPC). Multilineage directed differentiation efficiently produced functional cortical neurons, cardiomyocytes and hepatocytes. Pilot users further demonstrated line versatility by generating kidney organoids, T-lymphocytes and sensory neurons. A frameshift knockout was introduced into MYBPC3 and these cardiomyocytes exhibited the expected hypertrophic phenotype. Whole genome sequencing (WGS) based annotation of PGPC lines revealed on average 20 coding variants. Importantly, nearly all annotated PGPC and HipSci lines harboured at least one pre-existing or acquired variant with cardiac, neurological or other disease associations. Overall, PGPC lines were efficiently differentiated by multiple users into cell types found in six tissues for disease modeling, and clinical annotation highlighted variant-preferred lines for use as unaffected controls in specific disease settings.

developmental biology↗

bioRxiv2018

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Oligomerization of RIG-I-like receptors on cytosolic dsRNA nucleates self-assembly of the mitochondrial antiviral signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

molecular biology↗