Genes to Cells

BackgroundHeterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty and Orly that are transcribed in testis and have known functions in male fertility. Of these Ssty and Sly encode proteins - yet all the transcripts are not translated. To investigate the roles of these Y-heterochromatic transcripts further, we analyzed them. MethodsMice with 2/3rd deletion of the Y-chromosome (XYRIIIqdel) and its wild type (XYRIII) were used in this study. Bioinformatic approaches, small RNA northern blots, Electrophoretic Mobility Shift Assays, Luciferase reporter assays, dPCR analysis, RT-qPCR assays and western blotting techniques were used to identify piRNAs that regulate autosomal genes. ResultsWe demonstrate that the multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. We observed sequences homologous to these piRNAs in the UTRs of a few autosomal genes, which are differentially expressed in the sperms of XYRIIIqdel mice. Furthermore, the Endogenous Retrovirus Element (ERV) LTR, found in the Orly1 transcript identified piRNAs in the database, showed homology to UTRs and associated genomic regions of a few autosomal genes. Orly1 showed a reduction in genomic copy number by digital PCR in XYRIIIqdel mice. One of the four autosomal genes containing the ERV segment in their UTRs, showed a differential testicular protein expression in the mutant mice. ConclusionsThus, we further elucidate that different classes of repeats from Y-chromosome regulate autosomal gene expression via piRNAs. Besides, this study also identified novel roles for a Y-derived ERV in autosomal gene regulation in testis.

Turbot (Scophthalmus maximus) is an important aquaculture species, but the genomic organization and expressed diversity of its antibody repertoire remain incompletely characterized. In this study, we annotated the immunoglobulin heavy chain (IGH) locus using the haplotype resolved fScoMax1.1 genome assembly, and we used this as a reference to profile the expressed turbot IgM, IgD and IgT repertoires in skin and spleen. The primary IGH locus was located on chromosome 19, spanned approximately 72 kb, and contained 25 IGHV genes, including 24 functional genes and one pseudogene, together with three IGHD, seven IGHJ and three IGHC genes corresponding to IgT, IgM and IgD. Comparison with the alternate fScoMax1.1 haplotype and a second turbot genome assembly showed conserved IGHD, IGHJ and IGHC content, whereas IGHV gene number differed among assemblies. High throughput 5RACE repertoire sequencing revealed isotype and tissue associated differences in expressed IGH diversity. IgM represented the dominant productive repertoire in both skin and spleen and showed the highest clonotypic diversity, particularly in spleen. IgD displayed an intermediate profile, whereas IgT was more enriched in skin and exhibited the strongest clonal restriction. IGHV subgroup usage was dominated by IGHV3 in IgM and IgD, whereas IgT showed a distinct profile characterized by preferential use of IGHV4, especially in skin. Gene level analysis further showed broad IGHV-IGHJ pairing in IgM and IgD, with preferential use IGHJ3 segment, while IgT sequences paired exclusively with IGHJT. Clonotype sharing between skin and spleen was isotype dependent, being strongest for IgT, intermediate for IgM, and negligible for IgD, suggesting that clonal expansion did not necessarily predict inter tissue trafficking. Together, these results provide a curated genomic and expressed repertoire framework for turbot IGH genes and reveal isotype specific organization of antibody diversity, with IgT displaying a particular repertoire pattern.

Objective To develop and validate a risk model for predicting postoperative bleeding in patients with thyroid cancer. Methods A total of 2800 consecutive patients diagnosed with thyroid cancer in the Department of Thyroid and Breast Surgery of the Affiliated Hospital of Xuzhou Medical University between January 2020 and December 2023 were retrospectively analyzed. Patients were categorized into two groups based on postoperative bleeding occurrence: bleeding and non-bleeding groups. Univariate and multivariate logistic regression analyses were utilized to screen independent risk factors. Meanwhile, risk prediction models were developed and nomogram . Subgroup analysis was performed to identify independent risk factors. The predictive effects of the models were assessed using the Hosmer-Lemeshow test and receiver operating characteristic (ROC) curves. Results Of the 2800 recruited patients, 50 had postoperative bleeding, with an incidence rate of 1.7%. Multivariate logistic regression analysis showed that age, hypertension, total thyroidectomy, tumor size [&ge;]4 cm, and operation time [&ge;]90 min were the risk factors for postoperative bleeding in thyroid cancer patients (P<0.05). A risk prediction model was established based on the above factors, and the area under the ROC curve was 0.881, with a sensitivity of 94.0%, a specificity of 67.3%, and an accuracy of 74.0%. Decision curve analysis revealed that the model had good predictive ability. Conclusions The constructed risk prediction model has good predictive power and can provide a reference for healthcare professionals to predict the risk of bleeding in patients after thyroid cancer surgery.

The plasmodium of Myxogastoria (a group of amoeboid protists) species often crawls around the forest floor to feed while searching for places to form fruiting bodies for reproduction (sporulation). Certain environmental factors that trigger sporulation have been reported; however, other unknown factors are also expected. In this study, we reported field observations of Physarum rigidum and Fuligo septica. Inspired by the field observation, we examined the effects of multiple factors on sporulation in laboratory experiments using Physarum polycephalum. We found that:(1) there was a critical body size below which sporulation did not occur under our experimental conditions and (2) the plasmodium selected its sporulation sites from the available landscape of the experimental arena: dry and low sites for the majority and dry and high sites for the minority. Further analysis revealed that they preferred the edge area at the high site. We discuss the possible ecological importance of the threshold and location preference

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that undergoes lytic replication in epithelial and other permissive cell types and can establish latency in peripheral neurons. ICP22 is a multifunctional HSV-1 immediate-early protein that localizes to the nucleus of infected cells; however, its interactions with host cellular factors remain incompletely understood. Here, ICP22 was demonstrated to interact with the human antisense function 1 protein (ASF1), including both ASF1a and ASF1b, in transfected cells and HSV-1-infected cells, respectively. ICP22 also colocalized with ASF1 in the nucleus. ICP22 amino acids 213 to 340 are important for the interaction of ICP22 with ASF1, whereas amino acids 37 to 153 of ASF1a and ASF1b are critical for their interactions with ICP22. Furthermore, ICP22 expression was associated with reduced ASF1-H3.1 co-immunoprecipitation under the tested conditions. ASF1 knockdown also reduced HSV-1-BAC-Luc luciferase output, indicating that ASF1 contributes to efficient infection-associated reporter activity in this study. Collectively, these results indicate that the interaction of HSV-1 ICP22 with ASF1 might help regulate the transcription of viral or cellular genes during HSV-1 infection. Keywords: HSV-1, ICP22, ASF1, histone H3.

The IFN-I (type I interferon) signaling pathway is the first line of defence against foreign pathogens. Stringent control of signalling pathways is necessary to maintain host immune responses and homeostasis. However, the underlying mechanism for its tight regulation is yet completely understood. In this study, we demonstrated that the TRIM family protein tripartite motif-containing 52 (TRIM52) is a novel negative regulator of IFN-{beta} production. Ectopically expressed TRIM52 markedly inhibited the activation of the IFN-{beta} promoter by ectopic expression of cGAS/STING, RIG-IN, or TRIF, MAVS, STING, and TBK1 but not by IRF3/5D, indicating that TRIM52 targets TBK1. TRIM52 also significantly inhibited the IFN-{beta}, ISG54, and ISG56 production, the dimerization of IRF3 and the nuclear localization of IRF3-YFP induced by ectopic expression of TBK1. Co-immunoprecipitation experiment revealed that TRIM52 specifically interacted with TBK1. Furthermore, the TBK1 protein, but not its mRNA, decreased considerably with increasing expression of TRIM52, and TRIM52 did not decrease the expression of the cGAS, STING, or IRF3 proteins. In addition, proteasome inhibitor MG-132 blocked the reduced TBK1 induced by TRIM52, indicating that TRIM52 caused TBK1 degradation via the proteasome pathway. Co-IP and ubiquitination assays demonstrated that TRIM52 promotion of K48-linked ubiquitination of TBK1, which depends on its E3 ubiquitin ligase. Collectively, our findings identify a previously unrecognized role of TRIM52 in regulating the IFN-I signalling pathway through targeting TBK1 for polyubiquitination and degradation.

The preparation of multi panel figures remains a labor intensive step in scientific publication. Albeit there are specific tools available to solve this problem, they are often highly specialized, difficult to install, or time consuming to learn. Griphus is a standalone graphical application designed for rapid composition and experimentation with multi panel figures, developed by and for zoological taxonomists. Functions specifically designed for multi panel composition include automatic figure numbering and placement, aspect ratio operations, spacers, layout rotation, layout suggestions, and automatic generation of figure legends, including scale bar descriptions. The software can perform both spatial interpretation of images on the canvas and work with a simple, editable layout formula. It also enables instant multi panel composition, with numbered images and automatic contrast selection for the numbers, obtained simply by loading images. User defined parameters such as target printable dimensions, resolution, spacing, and color mode are preserved throughout the work. The program produces coordinated outputs consisting of the final composite figure, a readable file describing the layout structure, and a .gri file storing images, transformations, and parameters for exact regeneration. Griphus is intended as a complementary tool to professional image software, providing a simple and efficient environment for constructing high quality multi panel figures.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

Microtubules are cytoskeletal polymers that play essential roles in eukaryotic cells, including structural support, cell division, and intracellular transport. During intracellular transport, kinesin motor proteins move cargo along microtubule tracks via their processive stepping. Recent studies have shown that the kinesin-1 KIF5C can damage the microtubule lattice while stepping. Microtubule damage can be repaired through incorporation of new tubulin subunits, however, excessive lattice damage results in microtubule breakage and disassembly. To identify cellular factors involved in microtubule repair, we performed an siRNA screen targeting microtubule-associated proteins (MAPs) known to regulate microtubule dynamics and stability. Based on the results, we investigated whether the end binding protein EB1 and cytoplasmic linker-associated protein 2 (CLASP2) contribute to repair of microtubule damage. To test this, we used a microtubule destruction assay in which damage was induced in microtubules gliding over surfaces coated with wild-type or mutant KIF5C proteins. Our findings suggest that CLASP2 directly facilitates microtubule repair, whereas EB1 does not. We further examined CLASP function using a microtubule repair assay and found that CLASP2 promotes repair by enhancing tubulin incorporation and reducing microtubule breakage. Together, these findings demonstrate that CLASP proteins play an important role in repairing and protecting against lattice damage caused by kinesin-1 motor activity. Our results further suggest that MAPs can directly regulate microtubule lattice integrity under mechanical stress generated by motor protein-driven intracellular transport.

Bacterial survival after ultraviolet (UV) exposure is shaped not only by the extent of DNA damage but also by the physiological state-dependent capacity for DNA repair. Here, we examined the mechanisms underlying growth phase-dependent UV resistance in Escherichia coli K-12 exposed to 262 nm UV irradiation. Stationary-phase cells required higher UV fluence for log inactivation than exponential-phase cells, whereas the levels of UV-induced DNA damage, assessed by cyclobutane pyrimidine dimer staining and real-time PCR, did not differ markedly between the two growth phases. Deletion of nucleotide excision repair (NER) genes, including uvrA, uvrB, uvrC, and uvrD, markedly reduced survival after UV irradiation, indicating that NER is essential for the high UV resistance of stationary-phase cells. Quantitative real-time reverse transcription PCR showed stronger UV-induced expression of several DNA repair and UV resistance genes, including uvrA, uvrB, cho, umuC, and umuD, in stationary-phase cells than in exponential-phase cells. Furthermore, deletion of the DNA cytosine methyltransferase gene dcm increased UV resistance and enhanced the expression of uvrB, cho, umuC, umuD, and sulA in stationary-phase cells. These findings suggest that DNA cytosine methylation modulates UV resistance in E. coli, at least in part by influencing NER- and SOS-associated gene expression.

Images in the electron microscope are formed by electron scattering and focusing. The spherical geometry of these processes gives rise to two coherent, conjugate spherical wave fronts, known as Ewald spheres. These spheres are associated with the two halves of the contrast transfer function (CTF), and their widths are determined by the focal gradient through the specimen. To properly correct for the CTF, each half of the CTF must be applied to an image individually and integrated into the reconstruction into the corresponding Ewald sphere. Theory indicates that this dual Ewald sphere reconstruction method should recover the maximal amount of information possible. This method was compared to the other reconstruction methods commonly used: the projection approximation (ignoring the Ewald sphere), the simple insertion and the single sideband methods. In simulated reconstructions the dual Ewald sphere method recovered the most information when the correct half of the CTF is matched to the corresponding Ewald sphere. If the wrong half is matched, the result worse than the projection approximation method. Examining reconstructions from real data indicated that the dual Ewald sphere method performs at least as well as the simple insertion method, but not as good as in simulations. The likely reason is the two-fold ambiguity in the assigned orientations of the particle images, which remains an issue to pursue in further studies. In conclusion, the dual Ewald sphere reconstruction method may offer the best way to calculate very high resolution reconstructions when the micrograph quality warrants it. HighlightsO_LIThe dual Ewald sphere reconstruction corrects for the two halves of the CTF. C_LIO_LIThe signs of the two halves of the CTF must correspond to the focal gradient. C_LIO_LIDetermining the focal gradient for individual particle images remains unresolved. C_LIO_LIComplex reconstructions indicate any real space phases are artifacts. C_LI

Whole-genome sequencing identifies focal DNA amplifications with base-pair resolution but cannot determine whether amplified sequences reside on extrachromosomal DNA (ecDNA, also known as double minutes) or within chromosomally integrated homogeneously staining regions (HSRs). DNA fluorescence in situ hybridization (DNA-FISH) metaphase spreads remain the gold standard for distinguishing these amplification states at single-cell resolution. Here, we present a detailed protocol for DNA-FISH metaphase spreads using human cancer cell lines, encompassing cell culture, metaphase arrest, hypotonic treatment, fixation, chromosome spreading, fluorescent probe hybridization, and fluorescence imaging. The protocol incorporates intermediate quality-control steps to verify successful chromosome dispersion and optimize metaphase spread quality, making the workflow accessible to laboratories without specialized cytogenetics expertise. Results demonstrate clear visualization of ecDNA and HSR amplification states using locus-specific probes and illustrate common technical artifacts that can affect interpretation. This protocol provides a robust and reproducible approach for studying the structural organization of oncogene amplification in cancer cells.

Brydes whales (Balaenoptera edeni edeni) are nationally protected in China, and the waters around Weizhou Island in the Beibu Gulf support one of the countrys few regularly observed coastal groups. However, acoustic data for this population remain limited, and the potential effects of local vessel noise are poorly described. We conducted 16 vessel-based surveys around Weizhou Island and adjacent waters in January 2024 using a low-disturbance sailboat platform and passive acoustic recorders, with concurrent visual observations where possible. We identified 734 low-frequency signals classified as putative Brydes whale vocalizations and quantified their temporal and spectral parameters. Call duration was significantly negatively correlated with maximum frequency and center frequency, but not with minimum frequency or bandwidth. Comparisons with published records indicate that the recorded signals are most similar to vocalizations previously reported from juvenile Brydes whales or mother-calf pairs, although individual source attribution could not be confirmed. Speedboat passage significantly increased root-mean-square sound pressure levels, and the dominant noise band overlapped the frequency range of the recorded Brydes whale signals, indicating potential for acoustic masking. These results expand the bioacoustic baseline for Brydes whales in Chinese coastal waters and provide evidence relevant to the management of vessel activity and whale-watching tourism around Weizhou Island.

Long-distance movement in insects has crucial impacts on agriculture, human health, and biodiversity. Although it was long assumed that only large, specialist insects had the navigation capacity to support long-distance dispersal, recent studies have demonstrated that smaller insects, such as the tiny fruit fly Drosophila melanogaster, can maintain extended, straight paths while flying or walking. This raises the question of whether other Drosophila species possess the navigation capacity to support extended dispersal. Resolving this question is particularly important for Drosophila suzukii(spotted-wing drosophila), a potent pest species that causes enormous damage worldwide to ripe fruit and berries. Spotted-wing drosophila has been thought to lack a capacity for long-distance dispersal, as prior studies have estimated maximal daily dispersal distances of less than 90 m. We developed a system to continuously track the flight trajectories of magnetically tethered D. suzukii relative to a discrete, overhead LED that mimicked the sun. We found that flies maintained remarkably straight flight headings that varied unpredictably across individuals. Male and female D. suzukii exhibited a similar navigation capacity; both sexes responded to rotation of a discrete sun stimulus with compensatory turns to maintain a stable relative heading. Our results suggest that D. suzukiihas an underappreciated capacity for rapid, radial dispersal, which could exceed 250 m in 15 min. This capacity may contribute to the pest species' invasiveness and its reliable, annual re-establishment in seasonally intolerable climates. Our findings highlight the importance of developing area-wide, regional strategies to manage the impacts of D. suzukii.

Animals rely on a wide range of environmental signals, including celestial and terrestrial cues for navigation. While celestial cues, such as the sun, play a major role in maintaining a constant heading during long-distance migration and dispersal, terrestrial cues provide an animal with a short-range navigation system, ideal to pinpoint highly specific locations. In Monarch butterflies, the simulation of a terrestrial landmark, i.e. a vertical stripe, induces an attraction behavior (all animals head toward the stimulus) while a small green light spot, simulating the sun, elicits menotactic orientation (animals adopt individual-specific headings relative to the stimulus). However, the mechanisms underlying how the animal distinguishes between a stimulus as a terrestrial landmark versus a celestial cue remains unclear. To explore this, we tested non-migratory Monarch butterflies (Danaus plexippus) in a flight simulator. The inner surface of simulator was equipped with an area of LEDs, allowing to present different visual stimuli to the butterflies during tethered flight. By systematically manipulating the stimulus width, height, brightness, and elevation we found that Monarch butterflies exhibited attraction behavior to high contrast areas, like stripe edges. Menotactic behavior was not achieved by solely decreasing the stimulus to a small light spot but also required for the stimulus to be presented at higher elevation to be interpreted as a sun stimulus. These findings suggest that multiple parameters, inherently set by the butterflys navigation system, are critical to interpret a visual stimulus as celestial cue or terrestrial landmark, producing dynamic switches between different orientation strategies during navigation.

Animals balance staying in a favorable environment with exploring new ones. In C. elegans chemotaxis, the process by which worms migrate toward an attractant has been extensively studied. However, what happens after they reach it remains largely unexplored, partly because conventional assays immobilize worms at the point of arrival. Here, we quantitatively analyzed chemotactic behavior upon reaching an attractive odor source using an immobilization-free chemotaxis assay. We observed that 62% animals left the isoamyl alcohol region after initially approaching it, a behavior we termed "leaving behavior." Quantitative analysis revealed that leaving behavior represents a distinct locomotor state compared with free-moving, high-concentration odor avoidance, and approach behavior. To test whether leaving behavior is related to olfactory adaptation, we analyzed mutants in adaptation-related genes. The proportion of leaving behavior was significantly increased in egl-4 loss-of-function mutants compared with wild-type animals, whereas arr-1 mutants showed no significant difference. These results suggest that egl-4 negatively regulates leaving behavior, suggesting a role for this kinase in stabilizing post-arrival behavioral states beyond its known function in olfactory adaptation. Our findings indicate that chemotaxis involves dynamic behavioral transitions even after reaching an attractant, consistent with an exploration-exploitation trade-off framework.

A parasitic mite of honey bee brood (Tropilaelaps mercedesae), is spreading through populations of Apis mellifera honey bees in new regions and poses a major threat to honey bee health. Despite its clear threat, the biology of this mite is poorly understood, with gaps on such fundamental issues as how fast its populations can grow. This leaves the beekeeping world underprepared to plan for its arrival and management. In this study, we documented the growth of T. mercedesae populations in untreated A. mellifera colonies in Thailand and South Korea, and did the same for another parasitic mite (Varroa destructor) when possible. We found that the population growth of T. mercedesae was variable but could reach high levels (daily r of 0.010, 0.036, and 0.057), while the population growth of V. destructor (r = 0.021) matched previous estimates. Our results indicate that T. mercedesae populations can grow rapidly but they do not always attain this potential. Based on our results, humidity should be studied as a potential driver of population growth. If future work can reveal key drivers of T. mercedesae population growth, this would help predict infestations and help design management strategies that exploit the pest's biological vulnerabilities.

Mammal tails have long been recognized for their diversity of morphological form and function, however, there remains a substantial gap between the motivation to understand and emulate the various performance functions of the tail and what is known about tail anatomy. In this study, we were motivated to discover the anatomical foundations of the fast, whipping motions of the tail of the lesser Egyptian jerboa (Jaculus jaculus), which may aid in the quick changes of direction as the animal escapes from predators using ricochetal bipedal hopping. We employed microCT scans, dissections, and museum data to describe the musculoskeletal anatomy of the jerboa in comparison with the laboratory mouse (Mus musculus) and rat (Rattus norvegicus). While many aspects of tail anatomy are conserved across these species, the jerboa does possess unique characteristics such as an extremely long tail arising from caudal vertebral elongation, development of extensive dorsal musculature differentiated into lateral and medial components to increase points of skeletal attachment, and a novel anatomical feature - the bi-lobed cranial transverse process - which serves as a supernumerary dorsal tendon attachment site and possible brace to protect the ventral tendons and intrinsic muscles for a section of caudal vertebrae which likely experiences high mechanical stress.

Heterochromatin protein 1 (HP1 is an intrinsic component of heterochromatin domains where it is involved in a diverse set of functions including heterochromatin spreading and organization, chromatin compaction and transcriptional silencing. It has been suggested that HP1 functions through a phase separation mechanism, a process that has been observed in vitro in the presence of N-terminal phosphorylation, nucleic acids and nucleosome arrays. HP1 can also interact with numerous binding partners that contain a specific motif called an HP1 access code (HAC). HACs recognize and bind to an interface formed by the chromoshadow (CSD) domains in the HP1 homodimer, the functional form of the protein. It has been shown that some HP1 binding partners can enhance its phase separation ability while others disrupt the process. Here, we focus on the interactions between HP1 and three binding partners, namely the p150 subunit of the chromatin assembly factor 1 (CAF-1), the N-terminal domain of the lamin B receptor (LBR), and the mitotic protein Shugoshin 1 (Sgo1). Using phase separation assays, we show that CAF-1 prevents HP1 phase separation while LBR and Sgo1 enhance it. Binding assays, mutational studies, NMR spectroscopy and computational analysis allow us to dissect the contributions of the HAC motifs, the charge patterns of the binding partner sequences and the role of N-terminal phosphorylation on HP1 in condensate formation. Our results demonstrate that each binding partner uniquely balances these contributions to modulate the properties of HP1, while electrostatic interactions dominate the regulation of phosphorylated HP1. These results suggest that HP1 binding partners play an important role in the modulation of its properties and the regulation of its functions in distinct biological contexts.

Visual search is a critical component of many professions such as military operations, baggage screening, and radiology. Aided Target Recognition (AiTR) systems are designed to highlight potential threats across the operator visual field in real-time, directing attention and improving accuracy. However, these systems may impact search and, consequently, situational awareness by diverting attentional resources from non-highlighted, yet relevant, locations. Previous work suggests that scene gist is extracted within the first 250 ms of scene onset (Vo & Henderson, 2010). As such, this study examined whether a 250 ms AiTR onset delay could encourage a more even distribution of attention. Participants searched synthetically generated scenes and classified each person in the scene as armed or unarmed. Depending on their condition, participants either saw the scenes unaugmented (No AiTR condition), with AiTR highlights consisting of red bounding boxes around armed people and yellow boxes around unarmed (AiTR condition), or with AiTR highlights presented 250 ms post scene onset (Delayed AiTR condition). A surprise memory test of background objects presented in the search scenes was administered to all participants upon completion of the search task. As predicted and preregistered, results showed less overt attentional deployment to background information (anything other than the people themselves) in the AiTR condition compared to No AiTR , however, decreased overt attentional deployment was not seen in the Delayed AiTR group. A similar pattern was observed in the memory data (with the AiTR condition having a lower score than the No AiTR condition and the Delayed AiTR condition), this difference was not significant.