Biology subjects

McKenzie, D.

Publications and source records attributed to McKenzie, D..

13 recordsLinked to original sources

bioRxiv2026

GIGYF2/4EHP-Mediated Translational Attenuation Maintains Cellular Homeostasis Following Ionizing Radiation

Translational regulation is a critical component of the cellular response to environmental stress. Ionizing radiation (IR) exists naturally at low doses (e.g., cosmic rays and radioactive materials) but is applied at much higher doses in clinical settings, where accelerated photons (X-rays) and particle beams (protons, ions etc) are utilized for the treatment of cancer. While the effects of IR on DNA damage and cell cycle are well established, its impacts on cellular RNA metabolism remains less understood. In particular, the role of the mRNA translation machinery in shaping the early cellular response to IR is largely unexplored. Here, we demonstrate that IR induces an acute and persistent translational repression. This acute repression is independent of the mTOR and Integrated Stress Response pathways, which are known regulators of mRNA translation in response to environmental cues. Instead, we discovered that the translational repression is, at least partially, mediated by the GIGYF2/4EHP translational repressor complex. We show that GIGYF2/4EHP recruitment to the mRNAs upon IR exposure is driven by rapidly enhanced interactions with RNA-binding proteins such as ZFP36 and components of the miRNA-Induced Silencing Complex (miRISC) that are poised on their target mRNAs. Importantly, the presence of the GIGYF2/4EHP complex is required for the maintenance of proteostasis and cell viability following irradiation. Together, our results establish mRNA translational control as a key determinant of cellular response to IR and identify GIGYF2/4EHP as a critical component of this adaptive mechanism.

cell biology↗

bioRxiv2024

Solution-phase indexing by kinetic confinement enables rapid, simple, and instrument-free single cell transcriptional profiling

Existing tools for single cell genomics require complex physical frameworks for the indexing of cellular nucleic acids, including proprietary instrumentation, droplet emulsions, and laborious combinatorial indexing schemes. The complexity and cost of these tools significantly constrains the use of single cell technologies across basic and translational research. Here, we describe an instrument-free method that uses novel, bifunctional indexing reagents to deliver index sequences directly to single cells followed by a biophysical process known as Kinetic Confinement to perform high-fidelity indexing of target molecules across thousands of single cells simultaneously in single-tube, solution-phase reactions. Kinetic Confinement enables simple, fast, and flexible single cell experiments, and allows straightforward scaling to very large sample numbers. We anticipate that assays based on Kinetic Confinement will significantly expand the scope, use, and impact of single cell analysis across fundamental and applied research, as well as within therapeutic development and ultimately applied clinical diagnostics.

genomics↗

bioRxiv2024

A repository of Ogden syndrome (NAA10) and NAA15 patient derived iPSC lines and isogenic pairs by X-chromosome screening and genome-editing.

Amino-terminal (Nt-) acetylation (NTA) is a common protein modification, affecting 80% of cytosolic proteins in humans. The human essential gene, NAA10, encodes the enzyme NAA10, as the catalytic subunit for the N-terminal acetyltransferase A (NatA) complex, including the accessory protein, NAA15. The first human disease directly involving NAA10 was discovered in 2011, and it was named Ogden syndrome (OS), after the location of the first affected family residing in Ogden, Utah, USA. Since that time, other variants have been found in NAA10 and NAA15. Here we describe the generation of 31 iPSC lines, with 16 from females and 15 from males. This cohort includes CRISPR-mediated correction to the wild-type genotype in 4 male lines, along with editing one female line to generate homozygous wild-type or mutant clones. Following the monoclonalizaiton and screening for X-chromosome activation status in female lines, 3 additional pairs of female lines, in which either the wild type allele is on the active X chromosome (Xa) or the pathogenic variant allele is on Xa, have been generated. Subsets of this cohort have been successfully used to make cardiomyocytes and neural progenitor cells (NPCs). These cell lines are made available to the community via the NYSCF Repository.

genetics↗

bioRxiv2023

Fibronectin: a natural barrier against prion infection

A distinctive signature of the prion diseases is the accumulation of the pathogenic isoform of the prion protein, PrPSc, in the central nervous system of prion-affected humans and animals. PrPSc is also found in peripheral tissues, raising concerns about the potential transmission of pathogenic prions through human food supplies and posing a significant risk to public health. Although muscle tissues are considered to contain levels of low prion infectivity, it has been shown that myotubes in culture efficiently propagate PrPSc. Given the high consumption of muscle tissue, it is important to understand what factors could influence the establishment of a prion infection in muscle tissue. Here we used in vitro myotube cultures, differentiated from the C2C12 myoblast cell line (dC2C12), to identify factors affecting prion replication. A range of experimental conditions revealed that PrPSc is tightly associated with proteins found in the systemic extracellular matrix (ECM), mostly fibronectin (FN). The interaction of PrPSc with FN decreased prion infectivity, as determined by standard scrapie cell assay. Interestingly, the prion-resistant reserve cells in dC2C12 cultures displayed a FN-rich ECM while the prion-susceptible myotubes expressed FN at a low level. In agreement with the in vitro results, immunohistopathological analyses of tissues from sheep infected with natural scrapie demonstrated a prion susceptibility phenotype linked to an extracellular matrix with undetectable levels of FN. Conversely, PrPSc deposits were not observed in tissues expressing FN. These data indicate that extracellular FN may act as a natural barrier against prion replication and that the extracellular matrix composition may be a crucial feature determining prion tropism in different tissues. Author summaryPrion diseases are complex fatal neurodegenerative disorders caused by a misfolded form of the cellular protein PrPC (PrPSc). Due to the potential zoonotic transmission of these disorders through animal-based food intake, it is crucial to identify the tissues in which PrPSc can accumulate and what might influence its tropism. Animal muscle (or meat) and related food products are highly consumed, raising concern of the involvement of muscle cells in prion replication. Muscle tissue from prion-affected animals contains low levels of infectivity and PrPSc is mostly associated with nerve structures rather than myofibers, whereas C2C12 myotubes, a muscle-derived cell type, efficiently replicate prions in vitro and generate high levels of infectivity compared with other cell cultures. We demonstrate a fibronectin-mediated interference with prion infection in differentiated C2C12 cultures that correlates with the findings in tissues from naturally scrapie-infected animals. Our results suggest that extracellular matrix composition, specifically regarding the presence of fibronectin, might determine prion tropism and dissemination.

biochemistry↗

bioRxiv2022

Nanopore sequencing identifies a higher frequency and expanded spectrum of mitochondrial DNA deletion mutations in human aging

BackgroundMitochondrial DNA (mtDNA) deletion mutations cause many human diseases and are linked to age-induced mitochondrial dysfunction. Mapping the mutation spectrum and quantifying mtDNA deletion mutation frequency is challenging with next generation sequencing methods. We hypothesized that long-read sequencing of human mtDNA across the lifespan would detect a broader spectrum of mtDNA rearrangements and provide a more accurate measurement of their frequency. ResultsWe employed nanopore Cas9-targed sequencing (nCATS) to map and quantitate mtDNA deletion mutations and develop analyses that are fit-for-purpose. We analyzed total DNA from vastus lateralis muscle in 15 males ranging from 20 to 81 years of age and substantia nigra from three 20-year-old and three 79-year-old men. We found that mtDNA deletion mutations detected by nCATS increased exponentially with age and mapped to a wider region of the mitochondrial genome than previously reported. Using simulated data, we observed that large deletions are often reported as chimeric alignments. To address this, we developed two algorithms for deletion identification which yield consistent deletion mapping and identify both previously reported and novel mtDNA deletion breakpoints. The identified mtDNA deletion frequency measured by nCATS correlates strongly with chronological age and predicts the deletion frequency as measured by digital PCR approaches. In substantia nigra, we observed a similar frequency of age-related mtDNA deletions to those observed in muscle samples, but noted a distinct spectrum of deletion breakpoints. ConclusionsNCATS-mtDNA sequencing allows identification of mtDNA deletions on a single molecule level, characterizing the strong relationship between mtDNA deletion frequency and chronological aging.

genomics↗

bioRxiv2022

Photocycle characterization of a blue-orange cyanobacteriochrome from Synechococcus sp. PCC 7002

Cyanobacteria employ photoreceptors called cyanobacteriochromes (CBCRs) to sense the colour and intensity of light. The information extracted from the solar spectrum is used for adaptive responses such as optimizing photosynthesis, phototaxis and cell aggregation. cGMP-phosphodiesterase/adenlylate cyclase/FhlA (GAF) domains are the principal light sensors in cyanobacteriochromes. They contain a conjugated bilin chromophore and boast an impressive spectral diversity. Characterizing the spectral characteristics of GAF domains in model strains, such as Synechococcus sp. PCC 7002, can open new avenues for optogenetics and biotechnology. Based on sequence analysis we predicted several different GAF domains in this strain. The SynPCC7002_a0852 gene encodes a single GAF domain with two cysteine residues: one in the conserved 3 helix and one in the conserved DXCF motif. Spectral analysis of recombinant SynPCC7002_A0852 with phycocyanobilin (PCB) showed that the protein cycles between two states, Po and Pb, which absorb orange and blue light, respectively. Measurements of kinetics identified Po as the dark state of the protein. Acid-denaturation analysis suggested that the 15E isomer of PCB is bound in the (dark) Po state, whereas 15Z is bound the (photoproduct) Pb state. Site-directed mutagenesis and iodoacetamide treatments showed that Cys73 in the DXCF motif is essential for the conversion from Po to Pb. Future experiments dark-purified protein/chromophore versions are required to establish the sequence of events in the photocycle. In summary, SynPCC7002_A0852 enables orange/blue colour perception in Synechococcus sp. PCC 7002 as other CBRCs of this protein family but might contain the energetically higher chromophore isoform in its dark state. Such photocycle has previously been found in bathy bacteriophytochromes but not in CBCRs.

molecular biology↗

bioRxiv2022

Remdesivir does not affect mitochondrial DNA copy number or deletion mutation frequency in aged male rats

Remdesivir is a leading therapy in patients with moderate to severe coronavirus 2 (SARS-CoV-2) infection; the majority of whom are older individuals. Remdesivir is a nucleoside analog that incorporates into nascent viral RNA, inhibiting RNA-directed RNA polymerases, including that of SARS-CoV-2. Less is known about remdesivirs effects on mitochondria, particularly in older adults where mitochondria are known to be dysfunctional. Furthermore, its effect on age-induced mitochondrial mutations and copy number has not been previously studied. We hypothesized that remdesivir adversely affects mtDNA copy number and deletion mutation frequency in aged rodents. To test this hypothesis, 30-month-old male F333BNF1 rats were treated with remdesivir for three months. To determine if remdesivir adversely affects mtDNA, we measured copy number and mtDNA deletion frequency in rat hearts, kidneys, and skeletal muscles using digital PCR. We found no effects from three months of remdesivir treatment on mtDNA copy number or deletion mutation frequency in 33-month-old rats. For the 33-month-old control rats, the average mtDNA copy number per nucleus was 2567, 1100, and 1869 for heart, kidney, and quadriceps, respectively. MtDNA deletion mutation frequency was 2.6x10-4, 1.6x10-4 and 4.7x10-3for heart, kidney, and quadriceps, respectively. These data support the notion that remdesivir does not compromise mtDNA quality or quantity at old age in mammals. Future work should focus on examining additional tissues such as brain and liver, and extend testing to human clinical samples.

molecular biology↗

bioRxiv2022

The efficacy of EphA2 tyrosine phosphorylation increases with EphA2 oligomer size

The receptor tyrosine kinase (RTK) EphA2 is expressed in epithelial and endothelial cells and controls the assembly of cell-cell junctions. EphA2 has also been implicated in many diseases, including cancer. Unlike most RTKs, which signal predominantly as dimers, EphA2 readily forms higher order oligomers upon ligand binding. Here we investigated if a correlation exists between EphA2 signaling properties and the size of the EphA2 oligomers induced by multiple ligands, including the widely used ephrinA1-Fc ligand, the soluble monomeric m-ephrinA1, and novel engineered peptide ligands. We used Fluorescence Intensity Fluctuation (FIF) spectrometry to characterize the EphA2 oligomer populations induced by the different ligands. Interestingly, we found that different monomeric and dimeric ligands induce EphA2 oligomers with widely different size distributions. Comparison of FIF brightness distribution parameters and EphA2 signaling parameters reveals that the efficacy of EphA2 phosphorylation on tyrosine 588, which is indicative of receptor activation, correlates with EphA2 mean oligomer size. However, other characteristics, such as the efficacy of AKT inhibition and ligand bias coefficients, appear to be independent of EphA2 oligomer size. This work highlights the utility of FIF in RTK signaling research and demonstrates a quantitative correlation between the architecture of EphA2 signaling complexes and signaling features.

biophysics↗

bioRxiv2022

Assessment of Human Renal Transporter Based Drug-Drug Interactions Using Proximal Tubule Kidney-Chip

Study of renal transporters is crucial for understanding drug disposition and toxicity, and more importantly, predicting potential drug-drug interactions (DDIs). However, conventional in vitro models often fail to predict renal transporter activity and are not scalable to a predictive clinical outcome due to in vitro-in vivo discrepancy. Here, we successfully developed a human Proximal Tubule Kidney-Chip model that emulated in vivo renal physiology and function to assess renal transporter-based DDIs. Active and improved functionality of key renal transporters including p-glycoprotein (P-gp), multidrug and toxin extrusion (MATE) 1 and 2-K, organic anion transporter (OAT) 1 and 3, and organic cation transporter (OCT) 2 were demonstrated using appropriate probe substrates in Kidney-Chips compared to transwell controls. Moreover, comparative transcriptomic analysis revealed that key efflux and uptake transporters were expressed significantly higher in the Kidney-Chip compared to the transwell. Additionally, key parameters obtained from substrate-inhibitor interactions in the model were used to predict clinical DDIs as well as clearance values, which were closer to in vivo clearances. Overall, these results support that the human Proximal Tubule Kidney-Chip can reliably assess the role of human renal transporters in drug disposition and drug interactions, providing a critical tool to assess renal transport in vitro.

cell biology↗

bioRxiv2022

Pan-phylum In Silico Analyses of Nematode Endocannabinoid Signalling Systems Highlight Novel Opportunities for Parasite Drug Target Discovery

The endocannabinoid signalling (ECS) system is a complex lipid signalling pathway that modulates diverse physiological processes in both vertebrate and invertebrate systems. In nematodes, knowledge of endocannabinoid (EC) biology is derived primarily from the free-living model species Caenorhabditis elegans, where ECS has been linked to key aspects of nematode biology. The conservation and complexity of nematode ECS beyond C. elegans is largely uncharacterised, undermining the understanding of ECS biology in nematodes including species with key importance to human, veterinary and plant health. In this study we exploited publicly available omics datasets, in silico bioinformatics and phylogenetic analyses to examine the presence, conservation and life-stage expression profiles of EC-effectors across phylum Nematoda. Our data demonstrate that: (i) ECS is broadly conserved across phylum Nematoda, including in therapeutically and agriculturally relevant species; (ii) EC-effectors appear to display clade and lifestyle-specific conservation patterns; (iii) filarial species possess a reduced EC-effector complement; (iv) there are key differences between nematode and vertebrate EC-effectors; (v) life stage-, tissue- and sex-specific EC-effector expression profiles suggest a role for ECS in therapeutically relevant parasitic nematodes. These data also highlight putative novel targets for anthelmintic therapies. To our knowledge, this study represents the most comprehensive characterisation of ECS pathways in phylum Nematoda and inform our understanding of nematode ECS complexity. Fundamental knowledge of nematode ECS systems will seed follow-on functional studies in key nematode parasites to underpin novel drug target discovery efforts. CONTRIBUTION TO THE FIELDThis manuscript reports the in silico characterisation of endocannabinoid (EC) signalling pathways across the nematode phylum. The physiological relevance and therapeutic potential of EC signalling in higher organisms has received significant attention. In contrast much of our knowledge on EC signalling in nematodes has been derived from the free-living nematode Caenorhabditis elegans where the EC signalling system appears to play key roles in nematode biology and features GPCRs distinct from vertebrate cannabinoid receptors. Unfortunately, the configuration and broader biological significance of EC signalling pathways across the nematode phylum, including in parasites of agricultural, veterinary and medical significance, remains unknown. The in silico exploration of the nematode EC signalling system reported here will provide baseline data on novel neuronal signalling pathways to seed future drug target discovery pipelines for parasites.

neuroscience↗

bioRxiv2020

Prion protein lowering is a disease-modifying therapy across prion strains, disease stages, and endpoints

Lowering of prion protein (PrP) expression in the brain is a genetically validated therapeutic hypothesis in prion disease. We recently showed that antisense oligonucleotide (ASO)-mediated PrP suppression extends survival and delays disease onset in intracerebrally prion-infected mice in both prophylactic and delayed dosing paradigms. Here, we examine the efficacy of this therapeutic approach across diverse paradigms, varying the dose and dosing regimen, prion strain, treatment timepoint, and examining symptomatic, survival, and biomarker readouts. We recapitulate our previous findings with additional PrP-targeting ASOs, and demonstrate therapeutic benefit against four additional prion strains. We demonstrate that less than 25% PrP suppression is sufficient to extend survival and delay symptoms in a prophylactic paradigm. Rise in both neuroinflammation and neuronal injury markers can be reversed by a single dose of PrP-lowering ASO administered after the detection of pathological change. Chronic ASO-mediated suppression of PrP beginning at any time up to early signs of neuropathology confers benefit similar to constitutive heterozygous PrP knockout. Remarkably, even after emergence of frank symptoms including weight loss, a single treatment prolongs survival by months in a subset of animals. These results support ASO-mediated PrP lowering, and PrP-lowering therapeutics in general, as a promising path forward against prion disease.

neuroscience↗

bioRxiv2020

High-throughput screens of PAM-flexible Cas9 variants for gene knock-out and transcriptional modulation

A key limitation of the commonly-used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site, which reduces the number of accessible genomic loci. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG) but their activity has been measured at only a small number of endogenous sites. Here we devised a high-throughput Cas9 pooled competition screen to compare the performance of both PAM-flexible Cas9 variants and wild-type Cas9 at thousands of genomic loci and across 3 modalities (gene knock-out, transcriptional activation and suppression). We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cutting. Of the PAM-flexible variants, we found that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combined xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

bioengineering↗

bioRxiv2018

Disturbance macroecology: integrating disturbance ecology and macroecology in different-age post-fire stands of a closed-cone pine forest as a case study

Macroecological studies have generally restricted their scope to relatively steady-state systems, and as a result, how biodiversity and abundance metrics are expected to scale in disturbance-dependent ecosystems is unknown. We examine macroecological patterns in a fire-dependent forest of Bishop pine (Pinus muricata). We target two different-aged stands in a stand-replacing fire regime, one a characteristically mature stand with a diverse understory, and one more recently disturbed by a stand-replacing fire (17 years prior to measurement). We compare the stands using macroecological metrics of species richness, abundance and spatial distributions that are predicted by the Maximum Entropy Theory of Ecology (METE), an information-entropy based theory that has proven highly successful in predicting macroecological metrics across a wide variety of systems and taxa. Ecological patterns in the mature stand more closely match METE predictions than do data from the recently disturbed stand. This suggests METEs predictions are more robust in late-successional, slowly changing, or steady-state systems than those in rapid flux with respect to species composition, abundances, and organisms sizes. Our findings highlight the need for a macroecological theory that incorporates natural disturbance and other ecological perturbations into its predictive capabilities, because most natural systems are not in a steady state.

ecology↗