Biology subjects

Malik, A.

Publications and source records attributed to Malik, A..

At least 19 recordsLinked to original sources

bioRxiv2026

ChEBI-N - Enrichment Analysis with the ChEBI Ontology

Introduction: Biological and chemical ontologies are structured representations that can be used to map entities such as metabolites onto broader chemical and biological categories. Enrichment analysis applied to such ontologies identifies classes whose representation within a set deviates from an expected background distribution, helping to interpret complex metabolomics measurements. Objectives: This study aimed to develop a tool for ChEBI (Chemical Entities of Biological Interest) ontology-based enrichment analysis, named ChEBI-N (ChEBI eNrichment). It is a successor of the previous tool BiNChE, with new features that broaden its applicability. Methods: We reimplemented BiNChE and extended it to accept SMILES or InChI strings as well as ChEBI IDs. These broaden applicability to datasets with entities that are not yet present in ChEBI. We also constructed organism-specific background populations. Results: We demonstrate the tool on three published metabolomics datasets covering wine, cheese, and human plasma samples. ChEBI-N correctly highlighted relevant enriched classes in all three datasets, such as flavonoids in the wine dataset and fatty acid classes in the cheese dataset. Comparing organism-specific and full-ChEBI backgrounds shows that restricted backgrounds reduce the inflated significance of generic classes, although the most enriched classes remain largely consistent. Conclusion: ChEBI-N is an extended successor to BiNChE for ontology-based enrichment analysis of metabolomics data, offering a hypothesis-generating complement to existing metabolomics analysis pipelines. Availability: ChEBI-N is freely available at chebin.hastingslab.org or as a Python package (pypi.org/project/chebin), with source code at github.com/ontology-tools/chebin, archived at Zenodo (doi:10.5281/zenodo.23264219) and Software Heritage (swh:1:rev:133a41792704eb7f1db746f36a5ae97e9247b2b7).

bioinformatics↗

bioRxiv2026

The Genetic Landscape of Structural Variants in Aspergillus flavus using ONT and PacBio Sequencing

Genetic rearrangement in the absence of selection is poorly understood. Long-read, high-depth sequencing has enabled analysis of the genetic landscape of spontaneous structural variants. We have applied PacBio Revio sequencing to identify the major classes of genetic alterations during long-term vegetative growth in Aspergillus flavus. These classes include a) precise deletions frequently associated with the flanking region of homology, b) clusters of short deletions that are heavily enriched within centromeric and subtelomeric regions that are typically 94% A+T, and c) deletions and expansions within a subset of homopolymer tracts. We find 70 homopolymer tracts longer than 44 bp, dispersed throughout the genome in noncoding regions and unassociated with transposable elements. A broader analysis of third-generation sequenced datasets from fungi found within the Phylum Ascomycota showed a high but variable prevalence of homopolymeric tracts across species. The adventitious identification of a putative flbA mutation, responsible for an altered conidial phenotype, by the Deep Variant AI system also demonstrates the power of this approach. These studies show that high-depth, long-read sequencing can probe SNPs, indels, homopolymeric tracts, and large-scale rearrangements in an unbiased fashion, and demonstrate that short deletions and homopolymer tract expansions arise spontaneously in A. flavus and other filamentous fungi.

genomics↗

bioRxiv2026

Aquaporin-4 mislocalization from astrocyte endfeet prolongs survival in a prion-cerebral amyloid angiopathy model

Aquaporin 4 (AQP4) water channels are polarized to astrocytic endfeet at blood vessel interfaces, and lose polarity in vascular diseases, including stroke, chronic traumatic encephalopathy, and Alzheimers disease. AQP4 modulates water influx and efflux in the interstitial fluid, yet how AQP4 localization impacts cerebral amyloid angiopathy (CAA) remains poorly understood. Here we show that astrocytic end feet and AQP4 are displaced from amyloid-bearing vessels in a prion-CAA mouse model that expresses GPI-anchorless PrPC. Displacing AQP4 genetically through deleting alpha-syntrophin (Snta1-/-) led to a marked prolongation in survival, together with reduced microglial inflammation and C1q, in prion-CAA-affected mice. Additionally, synaptic structural proteins were better maintained. Finally, the level and distribution of prion aggregates were similar among the mice, indicating that prion conversion and spread was not affected. These results suggest that reducing AQP4 water channel function slows the decline in a vascular amyloid disease by reducing neuroinflammation.

neuroscience↗

bioRxiv2026

Crossmodal Expectations in Material Perception

Humans rely on multiple sensory modalities, such as vision, audition, and touch, to perceive materials in everyday life. Previous research shows that multisensory perception leads to facilitation, yet the mechanisms responsible for this facilitation remain poorly understood. One potential mechanism is crossmodal prediction, whereby input from one modality generates predictions about another. While substantial research on multisensory facilitation has focused on bottom-up processes, such as spatial, temporal, and semantic congruency, the role of crossmodal predictions, particularly in material perception, has received little attention. To address this gap, we conducted two experiments, a reaction time task and a material rating task, in which participants viewed computer-generated animations of familiar objects being dropped to the ground. The paradigm exploited the natural temporal structure of impact events: pre-impact visual appearance provides information about an objects material and therefore can generate expectations about the forthcoming impact sound. Critically, participants saw the event only until before the impact, after which the video was masked. Thus, vision and audition were temporally aligned but not presented concurrently, allowing us to isolate the influence of visually driven expectations on the incoming auditory information without a bottom-up conflict. In some trials, the sound matched the expected material, but in a subset, it was incongruent, violating expectations elicited by the preceding visual information. Across both experiments, participants took longer to respond on incongruent than congruent trials, suggesting increased processing demands. In the rating task, incongruent trials also shifted material judgments, such that ratings reflected a weighted combination of incoming auditory information and visually driven predictions, with large individual differences in relative cue weighting. These findings suggest that priors on material properties from one modality, specifically vision, not only establish high-level expectations within the modality about an objects future state, but also extend across modalities.

neuroscience↗

bioRxiv2026

Api5 Regulates Genomic Stability and Chemotherapy Resistance in Cancer

Api5 is elevated in a number of cancers and is associated with many hallmarks of cancer, including resistance to apoptosis, immune escape, stemness, chemotherapy resistance, high proliferation, and cell-cycle dysregulation. In this study, we identified the DNA and chromatin-binding activities of Api5 in tumorigenic cells, as well as its association with genomic instability and chemotherapy resistance. Knockdown of Api5 resulted in reduced nuclear volume, DNA content, and chromosome number, and increased sensitivity to DNA damage. The survival of Api5-knockdown cells decreased following UV and cisplatin treatments due to the accumulation of damaged DNA and inefficient nucleotide excision repair. Interestingly, Api5 knockdown cells also exhibited low pChk1 levels following UV damage. Further, we confirmed the chemotherapy resistance phenotype in cancers with elevated Api5 levels, demonstrating that xenograft tumours with Api5 knockdown responded better to cisplatin, with significant tumour regression. SummaryApoptosis inhibitor 5 (Api5) contributes to chemotherapy resistance by conferring a survival advantage and promoting efficient DNA repair following genotoxic stress through regulation of Chk1 activation.

Cancer Biology↗

bioRxiv2026

Striatal Cholinergic Interneurons Signal Aversion and Drive Avoidance

Seeking reward and avoiding punishment are essential for survival. However, how the brain learns aversive value and generates avoidance remains poorly understood. We investigate acetylcholine-releasing striatal cholinergic interneurons (ChIs), which exhibit prominent excitation to salient behavioral cues but have largely been postulated to play a permissive role in reinforcement learning. Using voltage imaging in mice performing aversive versus reward conditioning tasks, we reveal learning-mediated changes in both subthreshold membrane voltage and suprathreshold spiking of individual ChIs. Across tasks, ChIs exhibit stronger immediate excitation to conditioned cues predicting negative outcomes than positive outcomes. This valence-dependent response augmentation reflects a learning-induced network change specific to cues predicting negative outcomes. Furthermore, brief optogenetic silencing of this immediate excitation selectively impairs conditioned avoidance, but not conditioned approach. Together, these results reveal that ChIs actively signal evidence for aversion and play a critical role in driving avoidance, highlighting a cholinergic mechanism underlying aversive learning.

neuroscience↗

bioRxiv2026

Butyrate synergizes with glucose to promote anaerobic growth of Staphylococcus aureus via anaplerotic metabolism and stress response pathways

Short-chain fatty acids (SCFAs) like butyrate and propionate are abundant microbiota-derived metabolites that influence bacterial physiology in host-associated niches like the gastrointestinal tract. However, their effects on Staphylococcus aureus under varying nutritional conditions remain incompletely understood. Here we investigated how SCFAs interact with glucose or galactose to regulate anaerobic growth, biofilm formation, and global transcription in S. aureus. Both SCFAs inhibit anaerobic growth in a dose-dependent manner but biofilm was differentially affected, with butyrate promoting and propionate suppressing biofilm formation. Glucose and galactose alleviated SCFA-mediated growth inhibition, with glucose exerting the strongest effect. Notably, glucose enhanced butyrate-associated growth and biofilm formation beyond glucose alone, whereas galactose produced more modest effects. Enzymatic and genetic analyses indicated that SCFA-sugar biofilms contain proteins and extracellular DNA and involve VraSR-dependent regulation. Transcriptomic profiling revealed broad metabolic reprogramming, including induction of urease genes, amino acid biosynthesis, and stress response pathways. Synergistic effects between butyrate and glucose were partially dependent on anaplerotic metabolism via pyruvate carboxylase, linking oxaloacetate regeneration from pyruvate to S. aureus SCFA adaptation. Together these findings demonstrate that the nutritional environment dictates whether SCFAs impair S. aureus growth or reprogram its physiology, promoting metabolic adaptation and biofilm formation under sugar-replete conditions.

microbiology↗

bioRxiv2026

A synonymous mutation in MSMEG_4729 occurs at a high frequency in spontaneous D29-resistant mutants of Mycobacterium smegmatis

Compassionate use of mycobacteriophage therapy highlights the promising potential of phage therapy as an alternative treatment option for antibiotic-resistant infections when conventional treatments fail. However, realizing the full potential of phage therapy requires addressing key challenges, including host immune responses, the limited arsenal of therapeutically-useful mycobacteriophages, and the emergence of phage resistance. Dissecting the mechanisms of phage resistance is critical for ensuring the effectiveness and sustainability of phage therapy. In this study, we demonstrate that exposure to the lytic mycobacteriophage D29 triggers diverse genetic changes in Mycobacterium smegmatis. A synonymous mutation in MSMEG_4729 arises frequently but is insufficient to confer D29 resistance on its own. Instead, we identified possible Lsr2-independent activation of the lipooligosaccharide (LOS) biosynthesis cluster in a D29-resistant mutant harboring this mutation. We have also detected the possible activity of MSMEG_3213, a type II methyltransferase associated with m6A modifications in M. smegmatis. Finally, we isolated defense escape mutants (DEMs) of D29 capable of overcoming resistance in a strain with the MSMEG_4729 synonymous mutation. This profiling of M. smegmatiss likely defensive arsenal against the therapeutically-useful mycobacteriophage D29 provides a roadmap for further investigations and rational engineering of next-generation mycobacteriophages to combat drug-resistant mycobacterial infections. Impact statementInterest in phage therapy has been gaining traction recently, which is largely due to the serious threat of antimicrobial resistance. However, the efficacy and sustainability of phage therapy is threatened by certain challenges, which includes the ever existent threat of phage resistance. In this study, we identified several likely factors involved in D29 interaction with the model mycobacterium M. smegmatis. These findings set a roadmap for future investigations that would guide rational phage engineering to expand the currently limited arsenal of therapeutically useful mycobacteriophages as well as improve the efficiency of existing ones.

microbiology↗

bioRxiv2026

Likely role of promoter reconstitution in Mpr-mediated D29 resistance by Mycobacterium smegmatis

The multi-copy phage resistance gene (mpr) of M. smegmatis is a major factor in resistance to the lytic mycobacteriophage D29. Mpr is a membrane-bound exonuclease that cleaves phage DNA post injection, hence blocking downstream stages in the phage infection cycle. The mechanism of resistance allows for adsorption, is non-abortive and independent of any mutation in the gene. Rather, it depends on overexpression of a wild type copy of the gene. However, the underlying factor behind mpr overexpression in spontaneous D29-resistant mutants of M. smegmatis remained elusive. Here, we report that D29 infection triggers insertion sequence (IS) rearrangements, including the transposition and integration of IS6120 directly upstream of mpr. Mutants with IS6120 integration upstream of mpr show highly elevated Mpr expression. Whole genome sequence analysis reveals that IS6120 introduced a putative transcription factor-binding site and a canonical -35 promoter element at the integration site, hence reconstituting a fuller promoter (rcp) than the original promoter (wtp) at the site. Promoter reporter assays suggest that rcp is a far stronger promoter than wtp, implying that elevated mpr expression in D29-resistant mutants with this transposition event could be due to promoter reconstitution. While strains with this transposition event appear to grow normally, rcp-driven, vector-borne Mpr overexpression appears to be toxic as it barely allows for colony formation on agar plates. This study reports a previously unknown factor likely behind mpr regulation in M. smegmatis, adding to the existing knowledge of mycobacterial anti-phage defense mechanisms and guiding rational phage engineering efforts for therapeutic applications.

microbiology↗

bioRxiv2025

A division-associated envelope protein, MAB_2363, drives intrinsic resistance and virulence in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to conventional antibiotics, significantly limiting treatment options. Our previous studies established that MAB_2362 (SteA) is a key regulator of cell division that contributes to intrinsic resistance and virulence. Considering that SteA-like proteins often act alongside SteB counterparts, we hypothesized that the adjacent gene MAB_2363 encodes the corresponding SteB-like division regulator. In this study, we found that deletion of MAB_2363 significantly increased susceptibility to multiple antibiotics and disrupted cell wall permeability. Microscopy revealed striking cell division defects in the mutant, including elongated cell morphology and multiple septa. Subcellular localization of a GFP-MAB_2363 fusion protein demonstrated its enrichment at division septa, confirming its direct involvement in cell division. Deletion of MAB_2363 resulted in attenuated virulence, as evidenced by reduced bacterial survival in macrophages and murine infection models. To assess its functional relation with MAB_2362, we compared the single-deletion mutant of MAB_2363 with the single-deletion mutant of MAB_2362 and the double-deletion mutant of MAB_2362-MAB_2363. Notably, the phenotypes of the MAB_2363 mutant, including cell division defects, antibiotic susceptibility, and virulence, were markedly milder than those of the other two mutants. Collectively, these findings indicate that MAB_2363 functions as a secondary but essential division-associated factor that operates during cell division, thereby influencing intrinsic resistance and virulence in M. abscessus.

microbiology↗

bioRxiv2025

Macrophages drive a fibrogenic gene program of periductal fibroblasts in pediatric primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) is an autoimmune, cholestatic liver disease characterized by inflammation and fibrosis surrounding bile ducts. The cellular crosstalk driving periductal fibrosis remains poorly defined. This study applied a multi-omics approach integrating MERSCOPE spatial transcriptomics, bulk RNA-seq, and SomaScan proteomics to characterize fibrotic periductal regions and their cell-cell communications. Macrophages (MP) expressing moderate-to-high CD163 were found co-localized with cholangiocytes, T cells, and collagen-producing hepatic stellate cells (HSC). Cell niche analysis identified periductal regions with elevated fibrotic signals, in which cell-cell communication analysis revealed MP-HSC interactions involving 17 fibrotic driver genes in MP (e.g., IFNGR1, CSF1R, CD163) and six fibrotic effector genes in HSC (e.g., COL1A2, VCAN, MMP2). In validation analyses, bulk RNA-seq data showed higher driver and effector gene scores in PSC with established fibrosis compared to early-stage PSC and autoimmune hepatitis (AIH). Plasma proteins encoded by MP driver genes were elevated in autoimmune liver disease (AILD) and in patients with elevated ([≥]3.29 kPa) liver stiffness on MR elastography. These findings suggest that macrophages engage in localized crosstalk with HSC, activating fibrotic gene programs and contributing to periductal fibrosis in PSC, thereby identifying potential molecular targets for therapeutic intervention.

immunology↗

bioRxiv2025

Perceiving Material Qualities from Moving Contours

While research on the perception of line drawings has long demonstrated the importance of contours in object recognition, recent work shows that contours can also convey material properties. For example, even simple 2D shapes with varying contours have been shown to evoke vivid impressions of different materials (Pinna & Deiana, 2015). However, such static representations capture only a single moment in time. When a material moves, its contours shift, evolve, or deform over time, creating contour motion. Does this contour motion convey diagnostic information about material properties, independent of surface appearance? Existing studies on the role of dynamic cues in material perception either use fully rendered 3D stimuli, where contour motion is confounded with rich surface information, or motion-only displays (dynamic dot stimuli or noise patches), which eliminate surface cues but also lack clearly defined contours. As a result, the relative contribution of contour motion to material perception remains unclear. To address this gap, we measured how human observers perceive materials from dynamic line drawings ("line"), compared to animations of fully textured stimuli that carry optical and motion information ("full"), as well as dynamic dot stimuli ("dot"). Stimuli were rendered versions (full, dot, line) of material animations from five material categories (jelly, liquid, smoke, fabric, and rigid-breakable). In one experiment, participants rated five material attributes (dense, flexible, wobbly, fluid, airy motion), and in a second experiment, participants were asked to choose one of the two materials that is more similar to a third material across all possible combinations. Results from both experiments consistently reveal that 1) Dynamic line drawings vividly convey mechanical material properties, and 2) the similarity in material judgments between line and full conditions was larger than that between dot and full conditions. We conclude that contour motion carries rich information about mechanical material qualities.

neuroscience↗

bioRxiv2025

An ancient X chromosomal region harbours three genes potentially controlling sex determination in Cannabis sativa

O_LISex determination mechanisms in dioecious plants remain poorly understood yet offer an excellent model system to study genetic changes underlying morphological evolution. C_LIO_LIWe investigated the genetic basis of sex determination in Cannabis sativa, combining QTL mapping in a segregating population, comparative transcriptomics between monoecious and dioecious cultivars, and a genomic analysis of X-Y chromosome divergence. C_LIO_LIQTL mapping identified Monoecy1, a locus on the X chromosome putatively controlling the monoecy-dioecy trait. This locus resides in the most ancient and diverged region of the sex chromosomes and contains three genes within 60,000 bp (CsREM16, lncREM16, and CsKAN4) with distinct sex-specific and monoecy-specific expression patterns. C_LIO_LIMonoecy1 harbours genes for male-female as well as monoecious-dioecious sex determination. We propose that the combinatorial interaction of CSREM16, lncREM16 and CsKAN4 provides a unifying genetic framework for understanding male-female and monoecious-dioecious sex determination in Cannabis sativa. C_LI

plant biology↗

bioRxiv2025

Tolerance to Lung Infection in TWIK2 K+ Efflux Mediated Macrophage Trained Immunity

Lung macrophages such as alveolar macrophages (AM{varphi}) are essential for innate immune function in the lungs. It is now apparent that macrophages can be trained to become better at attacking infections. Although trained immunity is thought to result from metabolic and epigenetic reprogramming, the underlying mechanisms remain unclear. Here, we report that macrophages can be trained by extracellular ATP, which is ubiquitously released during inflammation. ATP ligates the canonical Purinergic Receptor 2 subtype X7 receptor (P2X7) to mediate endosomal Two-pore domain Weak Inwardly rectifying K+ channel 2 (TWIK2) translocation into the plasma membrane (PM). This enables the cells to transit to a ready state for microbial killing in two directions: first, K+ efflux via PM-TWIK2 induces NLRP3 inflammasome activation, which further activates metabolic pathways; second, upon bacterial phagocytosis, PM-TWIK2 internalizes into phagosome membrane with proper topological orientation, where TWIK2 mediates K+ influx into phagosomes to control pH and ionic strength favoring bacterial killing. Therefore, the enhanced association of TWIK2 in phagosomal and plasma membranes signaled by danger-associated molecular patterns (DAMPs), such as ATP, mediates trained immunity in macrophages and enhances microbiocidal activity.

immunology↗

bioRxiv2025

A simple and reliable PCR-based method to differentiate between XX and XY sex genotypes in Cannabis sativa

Cannabis sativa is mainly a dioecious plant, which means that female and male flowers develop on separate individuals, which is controlled by an XY sex determination system: females have two X chromosomes and male plants carry an X and a Y chromosome. C. sativa is a crop that has a wide variety of applications, some of which depend on the sex of the plant. Females are, for example, used for the cannabinoid production, as cannabinoids are produced in the female inflorescence. However, while adult C. sativa individuals present high sexual dimorphism, it is not possible to phenotypically distinguish male and female plants before flowering. Here we present the identification of a highly conserved sex marker, CsPDS5, and the development of a robust, reliable and affordable PCR-based method to determine the sex genotype. In contrast to other sex genotyping methods, our approach relies on a gene polymorphic between the X and Y chromosomes and therefore requires only a single PCR with one pair of primers. The method was tested in 14 cultivars and 6 crossings, with different tissues and developmental stages, on a total of 600 samples, with 100% accuracy. Our assay allows early sex identification and hemp selection, which is useful for both research and industrial purposes. Finally, the pipeline presented here to identify genes polymorphic between the X and Y chromosomes can serve to discover new sex markers, not only in C. sativa, but other dioecious plants and other organisms with 2 sexes.

plant biology↗

bioRxiv2025

p70S6 kinase-dependent phosphorylation of the μ2 subunit of the AP2 adaptor complex is needed for clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) internalizes cell-surface receptors via clathrin-coated invaginations of the plasma membrane. Both clathrin and endocytic cargo are recruited to these sites by the adaptor protein complex AP2. AP2 cycles between a closed cytoplasmic conformation and an open membrane-bound state, and efficient CME requires both conformations and their dynamic interconversion. The mechanisms regulating these conformational changes, which include post-translational modifications of the AP2, remain incompletely understood. Here, we report that p70S6 kinase phosphorylates the {micro}2 subunit of the AP2 and that the phosphorylation of serine 45 (S45) depends on p70S6K activity. Loss of S45-{micro}2 phosphorylation results in decreased internalization of canonical CME cargo such as transferrin and PDGF receptors. In Caenorhabditis elegans, lack of S45-{micro}2 phosphorylation produces directionally similar but markedly weaker phenotypes than AP2 loss of function. Live imaging and in silico dynamic modelling suggest that S45-2 phosphorylation has impact on the conformational changes of the AP2 complex. These findings identify a p70S6K-dependent mechanism that modulates AP2 function and further strengthen the importance of post-translational regulation in controlling CME. Summary statementClathrin-mediated endocytosis is essential for receptor internalization and cell signaling. This study identifies a novel regulatory mechanism in which p70S6K-mediated phosphorylation of the AP2 subunit 2 at Ser45 modulates AP2 conformational dynamics and facilitates receptor endocytosis.

cell biology↗

bioRxiv2025

A-beta-induced distress of astrocytes triggers Alzheimer disease pathology through non-canonical delta secretase activity

The importance of astrocytes for Alzheimer disease (AD) pathology is increasingly appreciated, yet the mechanisms whereby this cell type impacts neurodegenerative processes remain elusive. In a genetic mouse model with diminished astrocyte stress response, even low levels of amyloid-{beta} trigger astrocyte reactivity, resulting in brain inflammation and massive amyloid and tau pathologies. This dysfunctional response of astrocytes to amyloid-{beta} acts through activation of {delta} secretase, a stress-induced protease implicated in both amyloid and tau-related proteolytic processing. Our findings identify a failed astrocyte stress response to amyloid-{beta} as an early inducer of amyloid and tau co-morbidity, a noxious process in AD acting through a unique non-canonical secretase pathway.

neuroscience↗

bioRxiv2025

Clustering of Tandem Inverse Duplications in Centromeric and Subtelomeric Chromosomal Regions of Aspergillus flavus

The formation of inverted repeats frequently initiates rearrangements associated with gene amplification. Among these rearrangements are inverted triplications (TRP/INVs) that are associated with multiple disease states in humans. We have used the filamentous fungus Aspergillus flavus as a model system to analyze spontaneous DNA rearrangements using high-depth third-generation sequencing of DNA isolated from vegetatively cultured strains. Analysis of sequence data identified a class of infrequent and transient rearrangements having structures typical of TRP/INVs. These TRP/INVs form both unprocessed and processed species and have a variably sized deletion at the junction between direct and inverted sequences. We found that TRP/INVs are enriched in heterochromatic centromeric and subtelomeric A+T-rich regions, suggesting that this process is a source of genetic instability in these domains. Consistent with this finding, A+T-rich regions contain elevated levels of direct, inverted, and perfect palindromic repeats. Inverted junctions contain palindromic sequences that are typically associated with TRP/INVs. A closer examination of the class of genomic palindromes found at inverted junctions revealed a broad distribution, with the majority lying in AT-rich centromeric sequences. The distribution of TRP/INVs in centromeric and subtelomeric domains mirrors the frequency of palindromes, indicating that palindrome abundance is likely to be a driver of TRP/INVs. A further examination of predicted pairing patterns in palindrome-like structures suggests that these structures may function as sites of DNA resolution, strand transfer, and replicative stalling. These results are consistent with a replication-based model in which palindromes, under conditions of replication stress, facilitate the formation of TRP/INV structures. These studies also represent, to our knowledge, the first characterization of spontaneous TRP/INV formation, likely to underlie the complex rearrangements associated with higher eukaryotic genomic instability and disease states. AUTHOR SUMMARYInverted triplications (TRP/INVs), coupled with gene amplification, are sources of genetic instability. However, little is known about how these species arise spontaneously prior to selective pressures. We have established a fungal model system that uses long-read third-generation sequencing to identify low-abundance rearrangements in the absence of an intentional selection. We identified and characterized multiple classes of TRP/INVs flanked by palindromic sequences. These rearrangements were concentrated in the AT-rich centromeric and subtelomeric regions, suggesting that these sites are regulated recombinational hotspots. Palindromes that are also associated with TRP/INVs are also most prevalent in A+T-rich centromeric regions, suggesting that the abundance of palindromes, both as a source of DNA damage and fork stalling, is a driver of TRP/INV formation. These studies provide the first evidence (to our knowledge) of the spontaneous formation of TRP/INVs during DNA replication, which is likely to precede more complex sources of rearrangements associated with oncogenesis and other disease states in higher eukaryotes.

genetics↗