Biology subjects

Miller, M.

Publications and source records attributed to Miller, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Myeloid FtH Regulates Macrophage Response to Kidney Injury by Modulating Snca and Ferroptosis

This study explored the role of myeloid ferritin heavy chain (FtH) in coordinating kidney iron trafficking in health and disease. Synuclein- (Snca) was the sole iron-binding protein upregulated in response to myeloid FtH deletion (FtH{Delta}/{Delta}). Following kidney injury, FtH{Delta}/{Delta} mice showed worsened kidney function. Transcriptome analysis revealed coupling of FtH deficiency with ferroptosis activation, a regulated cell death associated with iron accumulation. Adverse effects of ferroptosis were evidenced by upregulation of ferroptosis-related genes, increased oxidative stress markers, and significant iron deposition in kidney tissues. This iron buildup in FtH{Delta}/{Delta} kidneys stemmed from macrophage reprogramming into an iron-recycling phenotype, driven by Spic induction. Mechanistically, we establish that monomeric Snca functions as a ferrireductase catalyst, intensifying oxidative stress and triggering ferroptosis. Additionally, Snca accumulates in kidney diseases distinguished by leukocyte expansion across species. These findings position myeloid FtH as a pivotal orchestrator of the FtH-Snca-Spic axis driving macrophage reprogramming and kidney injury. HighlightsO_LIMyeloid FtH deficiency drives kidney injury via activation of ferroptosis C_LIO_LIM{Phi} FtH deficiency induces Snca, linking iron dysregulation to M{Phi} function and response to kidney injury C_LIO_LIFerrireductase activity of monomeric Snca augments oxidative stress, promoting lipid peroxidation and ferroptosis C_LI In briefM{Phi} FtH modulates Snca and Spic to coordinate the injury response, linking iron trafficking to ferroptosis-induced kidney injury

pathology↗

bioRxiv2025

SVelfie: A method for discovering cancer drivers based on enrichment of likely functional structural variants

Structural variants (SVs) can drive tumorigenesis, yet discovering SV cancer drivers remains challenging1. Here, we present SVelfie (Structural Variants enriched with likely functional/impactful events), a statistical method to infer driver genes from SVs detected across a cohort of cancer genomes, based on enrichment of likely functional events. When testing SVelfie on lymphoma samples from the Pan-Cancer Analysis of Whole Genomes (PCAWG)2, it corroborates known tumor suppressor genes and also yields novel driver candidates.

bioinformatics↗

bioRxiv2025

A novel integrated framework to identify and characterize regional-scale pest insect dispersal

Forest pest insects cause major socio-economic impacts, global losses of millions of dollars, and ecosystem changes. A key challenge for their management is tracing regional dispersal events critical to outbreak dynamics. We developed an integrated tracing framework for pest insects by combining isotope geolocation, ecological data, and atmospheric modeling, and applied this framework to the eastern spruce budworm moth (Choristoneura fumiferana), the most severe defoliator of the North American boreal forest, to trace outbreak dispersal events. We first generated a North American model of bioavailable sulfur isotope ({delta}34S) variation in space (isoscape), which predominantly varied in response to oceanic sulfate deposition, and then calibrated it to spruce budworm tissues of known origin. We used an automated trap network with high temporal resolution to collect samples and identify potential immigration events of eastern spruce budworm to Nova Scotia, Canada. We traced the natal origin of these immigrants by integrating high-probability regions derived from{delta} 34S probabilistic assignments and HYSPLIT atmospheric dispersal models. Since high larval density is a strong predictor of budworm defoliation and emigration, HYSPLIT atmospheric dispersal models, which integrated spruce budworm behavioral constraints (e.g., flight velocity, altitude, and temperature thresholds), were started from defoliated areas to narrow-down the area of natal origin and estimate the migration route. We find that this integrated framework allows to narrow down the region of pest origins, restricting it to a few possible locations and demonstrating long-distance dispersal of spruce budworm across [~]400Km over the Gulf of St. Lawrence. Our framework demonstrates the utility of{delta} 34S geolocation in insect tracing, and that combining isotopic data with ecological indicators and atmospheric modeling offers an unprecedented resolution in understanding insect dispersal ecology. The approach is transferable to trace other migratory insect species to address conservation, agriculture, and bio-surveillance needs in the context of global environmental change.

ecology↗

bioRxiv2025

Single-cell multiome and spatial profiling reveals pancreas cell type-specific gene regulatory programs driving type 1 diabetes progression

Cell type-specific regulatory programs that drive type 1 diabetes (T1D) in the pancreas are poorly understood. Here we performed single nucleus multiomics and spatial transcriptomics in up to 32 non-diabetic (ND), autoantibody-positive (AAB+), and T1D pancreas donors. Genomic profiles from 853,005 cells mapped to 12 pancreatic cell types, including multiple exocrine sub- types. Beta, acinar, and other cell types, and related cellular niches, had altered abundance and gene activity in T1D progression, including distinct pathways altered in AAB+ compared to T1D. We identified epigenomic drivers of gene activity in T1D and AAB+ which, combined with genetic association, revealed causal pathways of T1D risk including antigen presentation in beta cells. Finally, single cell and spatial profiles together revealed widespread changes in cell-cell signaling in T1D including signals affecting beta cell regulation. Overall, these results revealed drivers of T1D progression in the pancreas, which form the basis for therapeutic targets for disease prevention.

genomics↗

bioRxiv2025

Local alkalinity enhancement using artificial substrates increases survivorship of early-stage coral recruits

Efforts to restore coral reefs using sexually derived coral recruits are often hindered by their low survivorship and growth, hence scalable interventions to improve these parameters are urgently needed. Here, we developed novel settlement substrates that modify the local chemical and hydrodynamic environments to provide local alkalinity enhancement (AE) within the laminar boundary layer to aid in coral restoration. Cement tiles with four different chemistries and two different surface topographies were tested in a novel flume system to quantify their ability to change local pH under reef-like conditions and their effect on larval settlement, survivorship, and growth of the endangered Caribbean coral, Orbicella faveolata. Chemistry had a minimal effect on the initial settlement of coral larvae, and textured tiles were preferred over smooth tiles. However, substrates that created a more alkaline local environment increased post-settlement survivorship. The increased survivorship of O. faveolata recruits on AE tiles was not due to increased growth on AE tiles, although trends in growth were dependent on chemistry and topography of tiles. Our results indicate that mixing sodium bicarbonate or sodium carbonate into cement used to fabricate artificial reef structures could be an effective means to enhance the development of coral cover. Significance StatementReef-building coral populations in South Florida and the Caribbean are nearing crisis, with low survivorship rates of new recruits being identified as a critical bottleneck limiting their recovery. Since ocean acidification is known to stress corals and hinder their growth, we hypothesized that enhancing substrate alkalinity could help mitigate ocean acidification effects by boosting coral growth and increasing survivorship during their critical early stages. This hypothesis was experimentally tested by adding sodium bicarbonate or sodium carbonate to settlement substrates and studying coral settlement preference, and coral spat growth and survival. Our results showed significantly higher survivorship on alkalinity enhanced substrates, indicating that this could be a promising restoration intervention.

ecology↗

bioRxiv2024

Heterogeneous responses to embryonic critical period perturbations among different components of the Drosophila larval locomotor circuit

As developing neural circuits become functional, they undergo a phase of heightened plasticity that facilitates network tuning in response to intrinsic and/or extrinsic stimuli. These developmental windows are termed critical periods (CPs), because perturbations during, but not outside, the CP can lead to lasting changes, such as the formation of sub-optimal or unstable networks. How separate, but connected elements, within a network might respond differently to a CP perturbation is not well understood. To study this, we used the locomotor network of the Drosophila larva as a model and heat stress as a CP stimulus that has ecological relevance. When embryos experienced heat stress the subsequent development of their locomotor network is changed, creating larvae with reduced crawling speed and decreased network stability. Developing body wall muscles and central neurons are sensitive to heat stress perturbations during distinct, consecutive phases of embryogenesis. Within the CNS, transient embryonic CP perturbation leads to increased synaptic drive from premotor interneurons to motoneurons, which in turn adopt reduced excitability. In contrast, the peripheral neuromuscular junction maintains normal synaptic transmission, despite significant structural changes. Thus, connected elements respond differentially to a CP perturbation, suggesting a sequence, or hierarchy, of network adjustment during the CP.

neuroscience↗

bioRxiv2024

Retinoic acid breakdown is required for proximodistal positional identity during amphibian limb regeneration

Regenerating limbs retain their proximodistal (PD) positional identity following amputation. This positional identity is genetically encoded by PD patterning genes that instruct blastema cells to regenerate the appropriate PD limb segment. Retinoic acid (RA) is known to specify proximal limb identity, but how RA signaling levels are established in the blastema is unknown. Here, we show that RA breakdown via CYP26B1 is essential for determining RA signaling levels within blastemas. CYP26B1 inhibition molecularly reprograms distal blastemas into a more proximal identity, phenocopying the effects of administering excess RA. We identify Shox as an RA-responsive gene that is differentially expressed between proximally and distally amputated limbs. Ablation of Shox results in shortened limbs with proximal skeletal elements that fail to initiate endochondral ossification. These results suggest that PD positional identity is determined by RA degradation and RA-responsive genes that regulate PD skeletal element formation during limb regeneration.

developmental biology↗

bioRxiv2024

Localized in vivo prodrug activation using radionuclides

Radionuclides used for imaging and therapy can show high molecular specificity in the body with appropriate targeting ligands. We hypothesized that local energy delivered by molecularly targeted radionuclides could chemically activate prodrugs at disease sites while avoiding activation in off-target sites of toxicity. As proof-of-principle, we tested whether this strategy of "RAdionuclide induced Drug Engagement for Release" (RAiDER) could locally deliver combined radiation and chemotherapy to maximize tumor cytotoxicity while minimizing exposure to activated chemotherapy in off-target sites. MethodsWe screened the ability of radionuclides to chemically activate a model radiation-activated prodrug consisting of the microtubule destabilizing monomethyl auristatin E caged by a radiation-responsive phenyl azide ("caged-MMAE") and interpreted experimental results using the radiobiology computational simulation suite TOPAS-nBio. RAiDER was evaluated in syngeneic mouse models of cancer using fibroblast activation protein inhibitor (FAPI) agents 99mTc-FAPI-34 and 177Lu-FAPI-04, the prostate-specific membrane antigen (PSMA) agent 177Lu-PSMA-617, combined with caged-MMAE or caged-exatecan. Biodistribution in mice, combined with clinical dosimetry, estimated the relationship between radiopharmaceutical uptake in patients and anticipated concentrations of activated prodrug using RAiDER. ResultsRAiDER efficiency varied by 250-fold across radionuclides (99mTc>177Lu>64Cu>68Ga>223Ra>18F), yielding up to 1.22{micro}M prodrug activation per Gy of exposure from 99mTc. Computational simulations implicated low-energy electron-mediated free radical formation as driving prodrug activation. Clinically relevant radionuclide concentrations chemically activated caged-MMAE restored its ability to destabilize microtubules and increased its cytotoxicity by up to 600-fold compared to non-irradiated prodrug. Mice treated with 99mTc-FAPI-34 and caged-MMAE accumulated up to 3000x greater concentrations of activated MMAE in tumors compared to other tissues. RAiDER with 99mTc-FAPI-34 or 177Lu-FAPI-04 delayed tumor growth, while monotherapies did not (P<0.03). Clinically-guided dosimetry suggests sufficient radiation doses can be delivered to activate therapeutically meaningful levels of prodrug. ConclusionThis proof-of-concept study shows that RAiDER is compatible with multiple radionuclides commonly used in nuclear medicine and has the potential to improve the efficacy of radiopharmaceutical therapies to treat cancer safely. RAiDER thus shows promise as an effective strategy to treat disseminated malignancies and broadens the capability of radiopharmaceuticals to trigger diverse biological and therapeutic responses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/606075v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@18aef37org.highwire.dtl.DTLVardef@5f36eforg.highwire.dtl.DTLVardef@10fa0ccorg.highwire.dtl.DTLVardef@105c416_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Single cell multiome profiling of pancreatic islets reveals physiological changes in cell type-specific regulation associated with diabetes risk

Physiological variability in pancreatic cell type gene regulation and the impact on diabetes risk is poorly understood. In this study we mapped gene regulation in pancreatic cell types using single cell multiomic (joint RNA-seq and ATAC-seq) profiling in 28 non-diabetic donors in combination with single cell data from 35 non-diabetic donors in the Human Pancreas Analysis Program. We identified widespread associations with age, sex, BMI, and HbA1c, where gene regulatory responses were highly cell type- and phenotype-specific. In beta cells, donor age associated with hypoxia, apoptosis, unfolded protein response, and external signal-dependent transcriptional regulators, while HbA1c associated with inflammatory responses and gender with chromatin organization. We identified 10.8K loci where genetic variants were QTLs for cis regulatory element (cRE) accessibility, including 20% with lineage- or cell type-specific effects which disrupted distinct transcription factor motifs. Type 2 diabetes and glycemic trait associated variants were enriched in both phenotype- and QTL-associated beta cell cREs, whereas type 1 diabetes showed limited enrichment. Variants at 226 diabetes and glycemic trait loci were QTLs in beta and other cell types, including 40 that were statistically colocalized, and annotating target genes of colocalized QTLs revealed genes with putatively novel roles in disease. Our findings reveal diverse responses of pancreatic cell types to phenotype and genotype in physiology, and identify pathways, networks, and genes through which physiology impacts diabetes risk.

genomics↗

bioRxiv2024

Hydrophilic/ Omniphobic droplet arrays for high-throughput and quantitative enzymology

Engineered enzymes with enhanced or novel functions are specific catalysts with wide-ranging applications in industry and medicine. Here, we introduce Droplet Array Microfluidic Enzyme Kinetics (DA-MEK), a high-throughput enzyme screening platform that combines water-in-air droplet microarrays formed on patterned superhydrophilic/omniphobic surfaces with cell-free protein synthesis to enable cost-effective expression and quantitative kinetic characterization of enzyme variants. By printing DNA templates encoding enzyme variants onto hydrophilic spots, stamping slides to add cell-free expression mix, and imaging the resulting arrays, we demonstrate reproducible expression of hundreds of enzyme variants per slide. Subsequent stamping of fluorogenic substrates and time-lapse imaging allows determination of Michaelis-Menten parameters for each variant, with measured catalytic efficiencies spanning 5 orders of magnitude and agreeing well with values obtained via traditional microtiter plate assays. DA-MEK consumes orders of magnitude less reagents than plate-based assays while providing accurate and detailed kinetic information for both beneficial and deleterious mutations. In future work, we anticipate DA-MEK will provide a powerful and versatile platform to accelerate enzyme engineering and enable screening of large variant libraries under diverse conditions.

bioengineering↗

bioRxiv2024

Multi-parametric thrombus profiling microfluidics detects intensified biomechanical thrombogenesis associated with hypertension and aging

Arterial thrombosis, which represents a critical complication of cardiovascular diseases, is a leading cause of death and disability worldwide with no effective bioassay for clinical prediction. As a symbolic feature of arterial thrombosis, severe stenosis in the blood vessel creates a high-shear, high-gradient flow environment that effectively facilitates platelet aggregation towards vessel occlusion even with platelet amplification loops inhibited. However, no approach is currently available to comprehensively characterize the size, composition and platelet activation status of thrombi forming under this biorheological condition. Here, we present a thrombus profiling assay that monitors the multi-dimensional attributes of thrombi forming in conditions mimicking the physiological scenario of arterial thrombosis. Using this platform, we demonstrate that different receptor-ligand interactions contribute distinctively to the composition and activation status of the thrombus. Our investigation into hypertensive and older individuals reveals intensified biomechanical thrombogenesis and multi-dimensional thrombus profile abnormalities, demonstrating a direct contribution of mechanobiology to arterial thrombosis and endorsing the diagnostic potential of the assay. Furthermore, we identify the hyperactivity of GPIb-integrin IIb{beta}3 mechanosensing axis as a molecular mechanism that contributes to hypertension-associated arterial thrombosis. By studying the interactions between anti-thrombotic inhibitors and hypertension, and the inter-individual variability in personal thrombus profiles, our work reveals a critical need for personalized anti-thrombotic drug selection that accommodates each patients pathological profile.

bioengineering↗

bioRxiv2024

A Novel Dual Probe-based Method for Mutation Detection using Isothermal Amplification

Cost efficient and rapid detection tools to detect mutations especially those linked to drug-resistance are important to address concerns of the rising multi-drug resistance infections. Here we integrated dual probes, namely a calibrator probe and an indicator probe, into isothermal amplification detection system. These two probes are designed to bind distinct regions on the same amplicon to determine the presence or absence of mutation. The calibrator probe signal is used as an internal signal calibrator for indicator probe which detects the presence or absence of the mutation. As an illustrative example, we evaluated the applicability of this dual probe method for detecting mutations associated with rifampicin (RIF) drug resistance at codons 516, 526 and 531 of the rpoB gene in Mycobacterium tuberculosis. In this assessment, we examined 127 artificial samples comprising wild type and mutant target sequences with single or multiple mutations. Our results demonstrated 100% accuracy of both wild type and mutant samples for mutations at codons 526 and 531. As regards to mutations at codon 516, the wild type was identified with 100% accuracy, while the mutant type was identified with 95% accuracy. Moreover, when we extended our evaluation using the Zeptometrix MTB Verification panel, our dual probe method correctly differentiated between the wild type and mutant, and identified the RIF-mutant strain which harbors mutations at codon 531 of the rpoB gene. Our isothermal mutation detection system, relying on dual probes exhibits a versatile approach. With the capability to identify mutations without prior knowledge of their specific mutation direction, our dual-probe method shows significant promise for applications in drug resistance nucleic acid testing, particularly in resource-limited settings.

molecular biology↗

bioRxiv2024

Next Generation Neuropeptide Y Receptor Small Molecule Agonists Inhibit Mosquito Biting Behavior

Female Aedes aegypti mosquitoes can spread disease-causing pathogens when they bite humans to obtain blood nutrients required for egg production. Following a complete blood meal, host-seeking is suppressed until eggs are laid. Neuropeptide Y-like Receptor 7 (NPYLR7) plays a role in endogenous host-seeking suppression and previous work identified small molecule NPYLR7 agonists that suppress host-seeking and blood feeding when fed to mosquitoes at high micromolar doses. Using structure activity relationship analysis and structure-guided design we synthesized 128 compounds with similarity to known NPYLR7 agonists. Although in vitro potency (EC50) was not strictly predictive of in vivo effect, we identified 3 compounds that suppressed blood feeding from a live host when fed to mosquitoes at a 1 M dose, a 100-fold improvement over the original reference compound. Exogenous activation of NPYLR7 represents an innovative vector control strategy to block mosquito biting behavior and prevent mosquito/human host interactions that lead to pathogen transmission.

pharmacology and toxicology↗

bioRxiv2023

Mu-opioid receptor knockout on Foxp2-expressing neurons leads to reduced aversion-resistant reward seeking

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic marker of intercalated (ITC) cells in the amygdala and a subset of striatal medium spiny neurons (MSNs), both of which express MORs in wild-type mice and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. For the current series of studies, we characterized the behavior of mice with genetic deletion of the MOR gene Oprm1 in Foxp2-expressing neurons (Foxp2-Cre/Oprm1fl/fl). Male and female Foxp2-Cre/Oprm1fl/fl mice were generated and heterozygous Cre+ (knockout) and homozygous Cre-(control) animals were tested for aversion-resistant alcohol consumption using an intermittent access (IA) task, operant responding for a sucrose reward, conditioned place aversion (CPA) to morphine withdrawal, and locomotor sensitization to morphine. The results demonstrate that deletion of MOR on Foxp2-expressing neurons renders mice more sensitive to quinine-adulterated ethanol (EtOH). Mice with the deletion (vs. Cre-controls) also consumed less alcohol during the final sessions of the IA task, responded less for sucrose under an FR3 schedule, and were less active at baseline and following morphine injection. Foxp2-MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2-expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking.

neuroscience↗

bioRxiv2023

A blood-brain-barrier penetrant AAV gene therapy rescues neurological deficits in mucolipidosis IV mice.

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits and progressive vision loss. Using AAV9 and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1-/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse that had exhibited significant degeneration at the time of the treatment. However, no increase of retinal thickness was observed after the gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies.

genetics↗

bioRxiv2023

Using a function-first "scout fragment"-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic scout fragments, which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

biochemistry↗

bioRxiv2023

Improved Protocol for Reproducible Human Cortical Organoids Reveals Early Alterations in Metabolism with MAPT Mutations

Human pluripotent stem cell (hPSC)-derived cortical organoids are powerful models but are often limited by low efficiency, variability, and stress-related artifacts. To address these challenges, we developed a scalable organoid platform with end-to-end quality control (QC) metrics spanning manufacturing and single-cell RNA-sequencing (scRNA-seq), developed using eight MAPT mutation isogenic line sets relevant to frontotemporal dementia (FTD-tau). Using a 96 slit-well format, we achieved [~]100% production efficiency across 64 lines. Controlled-release FGF2 enhanced iPSC pluripotency and reduced mesendodermal contaminants, while optimized SB431542 dosing enhanced cortical patterning across lines with variable TGFBR1/ALK5 expression. The resulting organoids displayed transcriptomic profiles and low-stress signatures closely aligned with the developing human cortex. Applying a cortical organoid scRNA-seq index (CortiCOSI), we identified early dysregulation of phosphatase regulators (PPP2CA, ANP32A) and the prefoldin subunit PFDN6 in MAPT V337M excitatory neurons before tau hyperphosphorylation and oligomerization. This platform improves scalability, reproducibility, and mechanistic insight in cortical organoid studies.

neuroscience↗

bioRxiv2023

Template switching between the leading and lagging strands at replication forks generates inverted copy number variants through hairpin-capped extrachromosomal DNA

Inherited and de novo copy number variants (CNVs) are increasingly found to be correlated with human developmental and cancerous phenotypes. Several models for template switching during replication have been proposed to explain the generation of these gross chromosomal rearrangements. We proposed a model of template switching (ODIRA--origin dependent inverted repeat amplification) in which simultaneous ligation of the leading and lagging strands at diverging replication forks could generate segmental inverted triplications through an extrachromosomal inverted circular intermediate. Here, we created a genetic assay using split-ura3 cassettes to trap the proposed inverted intermediate. However, instead of recovering circular inverted intermediates, we found inverted linear chromosomal fragments ending in native telomeres--suggesting that a template switch had occurred at the centromere-proximal fork of a replication bubble. As telomeric inverted hairpin fragments can also be created through double strand breaks we tested whether replication errors or repair of double stranded DNA breaks were the most likely initiating event. The results from CRISPR/Cas9 cleavage experiments and growth in the replication inhibitor hydroxyurea indicate that it is a replication error, not a double stranded break that creates the inverted junctions. Since inverted amplicons of the SUL1 gene occur during long-term growth in sulfate-limited chemostats, we sequenced evolved populations to look for evidence of linear intermediates formed by an error in replication. All of the data are compatible with a two-step version of the ODIRA model in which sequential template switching at short inverted repeats between the leading and lagging strands at replication forks generates inverted interstitial triplications. AUTHOR SUMMARYChromosomal rearrangements are a potent source of genetic variation in humans and other organisms. One specific type of rearrangement involves the increase in copies of segments of the genome. The variation in gene dosage that these rearrangements can cause has been associated with a wide range of neurological and other human disorders. A specific puzzling form of copy number increase consists of three tandem copies with the central copy in inverted orientation. How this rearrangement occurs is of great interest, yet the mechanisms responsible are only inferred by examining the sequence of final inverted products. Yeast provides a unique model system to explore the underlying molecular defects that give rise to inverted triplications. While the favored hypothesis suggests that double stranded DNA repair is the causative agent, we find that a particular form of template switching between strands at the replication fork, not a double stranded DNA break, is the initiating event. Using the awesome power of yeast genetics, we provide evidence in two different assays for this unique replication error that we call ODIRA (for Origin Dependent Inverted Repeat Amplification) and propose that it can also explain this form of copy number variant seen in human evolution and disease.

genomics↗