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Sullivan, W.

Publications and source records attributed to Sullivan, W..

15 recordsLinked to original sources

Decomposing scanner batch effects and their impact on pathology foundation models

Pathology foundation models (PFMs) are expected to represent the same tissue in the same way on any scanner, but their embeddings still carry scanner signals. Scanner-related image differences and the scanner sensitivity of PFMs have each been described. How tissue-dependent changes in color and spatial frequency relate to shifts in PFM representations, and what this means for correction, is less understood. We imaged 103 slides from 37 normal tissue types on six scanners (PanNormal; 95,780 matched locations) and used the real paired images as targets. We used the public PLISM dataset for external validation. First, we decomposed scanner differences into image properties. Color and spatial frequency changed together, by amounts that depended on tissue type. Second, corrections that matched these measured properties brought images closer to the target more reliably than representations. Pix2Pix reduced the image residual by 69% but increased UNI distance to the target by 27%. The same frequency matching helped one scanner and hurt another. Probes still distinguished corrected from real target embeddings after every correction. Third, changing one frequency band at a time showed that PFMs differ in how strongly each band moves them and whether it moves them toward the target. Across eight PFMs pretrained on whole-slide image tiles, the models that moved least under these small changes were the most robust to real scanner differences (Spearman {rho}=-0.90). Aligning images and aligning representations are therefore different goals. The gap between them depends on scanner, tissue, and model.

bioinformatics↗

Leaf movements as a quantitative metric for early stress detection

Early, precise, and non-destructive stress detection is essential for maintaining crop productivity, particularly in high-density plant growth systems like controlled environment agriculture (CEA), where manual monitoring is often impractical. Using plant motion as a proxy for growth and plant health, we demonstrate a method for early, non-invasive stress detection through quantitative leaf-movement analysis in lettuce and five other CEA relevant crops. Leaf-movement dynamics under stress were imaged with a low-cost, scalable Raspberry Pi imaging setup and quantified using a repurposed open-source motion estimation algorithm; Tracking Rhythms in Plants (TRiP). Our system detected stress-induced changes in leaf-movement within 1 hour of stress, with the timing dependent on the nature of the stress. Sustained reductions in leaf-movement coincide with decreased biomass accumulation. This approach offers a non-invasive, rapid, scalable, and cost-effective solution for continuous crop monitoring, with potential for application in both terrestrial and space farming CEA systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/732190v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@19ee20eorg.highwire.dtl.DTLVardef@b0804org.highwire.dtl.DTLVardef@3b3fa8org.highwire.dtl.DTLVardef@1d04026_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract:C_FLOATNO Quantification of leaf-movement dynamics as a high-throughput proxy for plant physiological status, enabling early stress detection and timely intervention to mitigate yield penalties in CEA settings (image made with biorender.org). C_FIG

plant biology↗

Wolbachia-induced cytoplasmic incompatibility produces heritable chromatin modifications that suppress position-effect variegation

Maternally transmitted Wolbachia often cause cytoplasmic incompatibility (CI), a sperm modification that kills host embryos lacking the endosymbiont. CI produces defects in paternal chromosome replication, condensation, and segregation during the first zygotic cell cycle, but a significant fraction of embryos progress normally through this and subsequent cycles and only exhibit defects at later developmental stages. These results, together with documented CI-induced epigenetic chromatin modifications, suggest heritable chromatin modifications are responsible for the developmentally delayed defects. Here, we conducted a Position-Effect Variegation (PEV) screen in Drosophila melanogaster using In(1)wm4to test for persistent effects on heterochromatin-mediated silencing in adults that survived CI. We show that Wolbachia acts as a variegation suppressor, or Su(var), increasing eye pigment when present in CI-inducing fathers, a reproducible effect observed across several maternal genotypes that differed in CI strength. That is, passage of the In(1)wm4through Wolbachia-infected males limits the spread of heterochromatin into the neighboring euchromatin in the progeny. This effect is consistent with disruption of heterochromatin establishment at the mid-blastula transition, when stochastic spreading of heterochromatin determines whether the displaced white gene is silenced. Surprisingly, maternal Wolbachia did not revert the PEV modification, and in one genotype, Wolbachia increased suppression. Together, our results demonstrate that Wolbachia-mediated chromatin effects persist to adulthood, are not corrected by CifA-dependent rescue, and can be compounded by maternal Wolbachia. These findings establish that rescue is incomplete at the level of heterochromatin-mediated silencing and suggest that CI-specific and constitutive Wolbachia chromatin effects may operate through at least partially independent pathways.

genetics↗

Wolbachia-induced Cytoplasmic Incompatibility drives epigenetic and maternally-influenced post-embryonic defects

A common form of Wolbachia-induced manipulation of host reproduction is Cytoplasmic Incompatibility (CI). In CI, Wolbachia modification of sperm results in pronounced defects in paternal chromosome condensation, replication, and segregation during the first mitotic division. Recent studies in D. simulans demonstrate that CI also induces independent and distinct later developmental defects resulting in high rates of mitotic errors during the mid-blastula transition and larval lethality. Here we show that in D. melanogaster, embryos derived from CI crosses experienced significant mitotic defects during gastrulation and increased larval lethality, both of which were eliminated in the progeny of Rescue crosses (both sexes infected). Examination of CI using females from 13 genetically distinct wild-type lines of the Drosophila Genetic Reference Panel (DGRP) revealed significant variation in the strength of the CI-induced lethality. Early embryonic pre-hatching and late larval lethal phases were uncorrelated, suggesting distinct factors influence the extent of the two lethal phases. Additionally, 3rd instar larvae and adults derived from D. melanogaster CI crosses exhibited locomotor defects that were also eliminated in Rescue crosses. These studies support a model in which Wolbachia effects on the sperm chromatin produce delayed developmental and locomotor defects, suggesting the involvement of epigenetic mechanisms. Support for this idea comes from our finding that levels of the heritable chromatin mark H3K27me1 are significantly elevated in CI-derived embryos. We conclude that the full measure of CI strength should take into account pre- and post-hatching lethality as well as locomotor defects. Together our findings suggest that the strength of these CI-induced phenotypes is governed at least in part by epigenetics and the maternal genetic background. AUTHOR SUMMARYSince the discovery of the antiviral properties of the bacteria Wolbachia, numerous strategies using this insect endosymbiont have been developed to combat vector-borne disease. While the success of these strategies relies on the rapid spread of Wolbachia through a naturally uninfected insect population, the molecular mechanisms by which Wolbachia promote their spread remain poorly defined. Current research on the primary mechanism behind Wolbachia spread, cytoplasmic incompatibility (CI), focuses on understanding the dramatic decrease in egg hatch rates that occurs when uninfected females mate with infected males. Here, we demonstrate that CI also induces substantial post-hatching larva and adult locomotor defects and lethality. In accord with these developmentally delayed defects, we show Wolbachia dramatically alter an epigenetic chromatin mark. Finally, we show that host maternal factors contribute to CI strength. Taken together, these results demonstrate that CI induces a much more expansive and developmentally delayed suite of phenotypes than previously reported.

microbiology↗

A patient-derived ovarian cancer organoid platform to study susceptibility to natural killer cells

Intratumoral heterogeneity drives therapy resistance and relapses in advanced stage cancers, such as ovarian cancer. Here, we present a live cell imaging assay using patient-derived ovarian cancer organoids for real time capture and quantification of natural killer cell-mediated apoptotic events in >500 organoids simultaneously. Our assay revealed significant inter- and intratumor response heterogeneity and identified a rare resistant organoid population, opening avenues to test immunomodulatory strategies that overcome resistance.

cancer biology↗

Fexinidazole and Corallopyronin A target Wolbachia-infected sheath cells present in filarial nematodes

The discovery of the endosymbiotic bacteria Wolbachia as an obligate symbiont of filarial nematodes has led to antibiotic-based treatments for filarial diseases. While lab and clinical studies have yielded promising results, recent animal studies reveal that Wolbachia levels may rebound following treatment with suboptimal doses of the antibiotic rifampicin. Previous work showed that a likely source of the bacterial rebound in females were dense clusters of Wolbachia in ovarian tissue. The number, size, and density of these Wolbachia clusters were not diminished despite antibiotic treatment. Here we define the cellular characteristics of the Wolbachia clusters in Brugia pahangi (wBp) and identify drugs that also target them. We have evidence that the Wolbachia clusters originate from newly formed sheath cells adjacent to the ovarian Distal Tip Cells. The dramatically enlarged volume of an infected sheath cell is strikingly similar to endosymbiont-induced bacteriocytes found in many insect species. Ultrastructural analysis reveals that the clustered Wolbachia present within the sheath cells exhibit a distinct morphology and form direct connections with the oocyte membrane and possibly the cytoplasm. This includes membrane-based channels providing a connection between Wolbachia-infected sheath cells and oocytes. We also determined that the Wolbachia within the sheath cells are either quiescent or replicating at a very low rate. Screens of known antibiotics and other drugs revealed that two drugs, Fexinidazole and Corallopyronin A, significantly reduced the number of clustered Wolbachia located within the sheath cells.

pathology↗

Acentric chromosome congression and alignment on the metaphase plate occur via kinetochore-independent forces

Chromosome congression and alignment on the metaphase plate involves lateral and microtubule plus-end interactions with the kinetochore. Here we take advantage of our ability to efficiently generate a GFP-marked acentric X chromosome fragment in Drosophila neuroblasts to identify forces acting on chromosome arms that drive congression and alignment. We find acentrics efficiently align on the metaphase plate, often more rapidly than kinetochore-bearing chromosomes. Unlike intact chromosomes, the paired sister acentrics oscillate as they move to and reside on the metaphase plate in a plane distinct and significantly further from the main mass of intact chromosomes. Consequently, at anaphase onset acentrics are oriented either parallel or perpendicular to the spindle. Parallel-oriented sisters separate by sliding while those oriented perpendicularly separate via unzipping. This oscillation, together with the fact that in monopolar spindles acentrics are rapidly shunted away from the poles, indicates that distributed plus-end directed forces are primarily responsible for acentric migration. This conclusion is supported by the observation that reduction of EB1 preferentially disrupts acentric alignment. In addition, reduction of Klp3a activity, a gene required for the establishment of pole-to-pole microtubules, preferentially disrupts acentric alignment. Taken together these studies suggest that plus-end forces mediated by the outer pole-to-pole microtubules are primarily responsible for acentric metaphase alignment. Surprisingly, we find that a small fraction of sister acentrics are anti-parallel aligned indicating that the kinetochore is required to ensure parallel alignment of sister chromatids. Finally, we find induction of acentric chromosome fragments results in a global reorganization of the congressed chromosomes into a torus configuration. Article SummaryThe kinetochore serves as a site for attaching microtubules and allows for successful alignment, separation, and segregation of replicated sister chromosomes during cell division. However, previous studies have revealed that sister chromosomes without kinetochores (acentrics) often align to the metaphase plate, undergo separation and segregation, and are properly transmitted to daughter cells. In this study, we discuss the forces acting on chromosomes, independent of the kinetochore, underlying their successful alignment in early mitosis.

cell biology↗

The Dietary Supplement Taurine Suppresses Ovarian Cancer Growth

Taurine, a non-proteogenic amino acid, and commonly used nutritional supplement can protect various tissues from degeneration associated with the action of the DNA-damaging chemotherapeutic agent cisplatin. Whether and how taurine protects human ovarian cancer (OC) cells from DNA damage caused by cisplatin is not well understood. We have found that OC ascites-derived cells contained significantly more intracellular taurine than cell cultures modeling OC. In culture, elevation of intracellular taurine concentration to OC ascites-cells-associated levels suppressed proliferation of various OC cell lines and patient-derived organoids, reduced glycolysis, and induced cell protection from cisplatin. Taurine cell protection was associated with decreased DNA damage in response to cisplatin. A combination of RNA sequencing, reverse phase protein arrays, live-cell microscopy, flow cytometry, and biochemical validation experiments provided evidence for taurine-mediated induction of mutant- or wild-type p53 binding to DNA, and activation of p53 effectors involved in negative regulation of the cell cycle (p21), and glycolysis (TIGAR). Paradoxically, taurines suppression of cell proliferation was associated with activation of pro-mitogenic signal transduction including ERK, mTOR, and increased mRNA expression of major DNA damage sensing molecules such as DNAPK, ATM and ATR. While inhibition of ERK or p53 did not interfere with taurines ability to protect cells from cisplatin, suppression of mTOR with Torin2, a clinically relevant inhibitor that also targets DNAPK and ATM/ATR, broke taurines cell protection. Our studies implicate that elevation of intracellular taurine could suppress cell growth, metabolism, and activate cell protective mechanisms involving mTOR and DNA damage sensing signal transduction.

cancer biology↗

Transpiration Responses to Potential Volatile Signals and Hydraulic Failure in Single Leaves of Vitis vinifera (cv. Shiraz) and Arabidopsis thaliana (Col 0) Utilising Sensitive Liquid Flow and Simultaneous Gas Exchange.

Volatile organic compounds (VOCs) may communicate stress between plants. However little appears to be documented on how such VOCs affect transpiration. Changes in transpiration in response to some VOCs was examined by measurement of flow (Q) at high resolution into detached leaves of Vitis vinifera (cv. Shiraz) and Arabidopsis (Col 0). Sensors recorded arrival and decay of volatiles at the leaf lamina. Moderate xylem tensions were developed in V. vinifera leaves by incorporating a hydraulic resistance in the flow pathway. Simultaneous recording of leaf gas exchange (Assimilation, A, and Transpiration, E) for both V. vinifera and Arabidopsis revealed that for Arabidopsis Q was stochastically restricted by the gas exchange cuvette but not E in the short term. Depending on the applied supply pressure cavitation could be controlled in V. vinifera evident from reduced Q, and leaf wilting. Stomatal closure occurred upon cavitation after a transitory increase in E and A, and after wilting began and was reversed by re-pressurization. VOCs were emitted from leaves corresponding to changes in flow rate, and light to dark transitions but not to cavitation. Volatile methanol but not ethanol or methyl salicylate induced a localised dose-dependent reversible stomatal closure in both V. vinifera and Arabidopsis.

plant biology↗

Distinct Wolbachia localization patterns in oocytes of diverse host species reveal multiple strategies of maternal transmission

A broad array of endosymbionts radiate through host populations via vertical transmission, yet much remains unknown concerning the cellular basis, diversity and routes underlying this transmission strategy. Here we address these issues, by examining the cellular distributions of Wolbachia strains that diverged up to 50 million years ago in the oocytes of 18 divergent Drosophila species. This analysis revealed three Wolbachia distribution patterns: 1) a tight clustering at the posterior pole plasm (the site of germline formation); 2) a concentration at the posterior pole plasm, but with a significant bacteria population distributed throughout the oocyte; 3) and a distribution throughout the oocyte, with none or very few located at the posterior pole plasm. Examination of this latter class indicates Wolbachia accesses the posterior pole plasm during the interval between late oogenesis and the blastoderm formation. We also find that one Wolbachia strain in this class concentrates in the posterior somatic follicle cells that encompass the pole plasm of the developing oocyte. In contrast, strains in which Wolbachia concentrate at the posterior pole plasm generally exhibit no or few Wolbachia in the follicle cells associated with the pole plasm. Taken together, these studies suggest that for some Drosophila species, Wolbachia invade the germline from neighboring somatic follicle cells. Phylogenomic analysis indicates that closely related Wolbachia strains tend to exhibit similar patterns of posterior localization, suggesting that specific localization strategies are a function of Wolbachia-associated factors. Previous studies revealed that endosymbionts rely on one of two distinct routes of vertical transmission: continuous maintenance in the germline (germline-to-germline) or a more circuitous route via the soma (germline-to-soma-to-germline). Here we provide compelling evidence that Wolbachia strains infecting Drosophila species maintain the diverse arrays of cellular mechanisms necessary for both of these distinct transmission routes. This characteristic may account for its ability to infect and spread globally through a vast range of host insect species.

cell biology↗

Wolbachia action in the sperm produces developmentally deferred chromosome segregation defects during the Drosophila mid-blastula transition

Wolbachia, a vertically transmitted endosymbiont infecting many insects, spreads rapidly through uninfected populations by a mechanism known as Cytoplasmic Incompatibility (CI). In CI, embryos from crosses between Wolbachia-infected males and uninfected females fail to develop due to the immediate action of Wolbachia-produced factors in the first zygotic division. In contrast, viable progeny are produced when the female parent is infected. Here, we find [~]1/3 of embryos from CI crosses in Drosophila simulans develop normally beyond the first and subsequent pre-blastoderm divisions. Developing CI-derived embryos then exhibit chromosome segregation errors during the mid-blastula transition and gastrulation. Single embryo PCR and whole genome sequencing reveal a large percentage of the developed CI-derived embryos bypass the first division defect. Using fluorescence in situ hybridization, we find increased chromosome segregation errors in gastrulating CI-derived embryos that had avoided the first division defect. Thus, Wolbachia in the sperm induces independent immediate and developmentally deferred defects. Like the initial immediate defect, the delayed defect is rescued through crosses to infected females.

cell biology↗

Kinetochore-independent sister chromosome separation is driven by plus-end microtubule dynamics

Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore-independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation. AUTHOR SUMMARYKinetochores, the site on the chromosomes to which microtubules attach driving the separation and segregation of replicated sister chromosomes, have been viewed as essential for proper cell division and accurate transmission of chromosomes into daughter cells. However previous studies demonstrated that sister chromosomes lacking kinetochores (acentrics) often undergo separation, segregation and transmission. Here we demonstrate that sister acentrics are held together through DNA intertwining. We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers. We found that acentric sister chromatids display unique patterns of separation using mechanisms independent of the kinetochore. Additionally, we identified the specific microtubule-associated proteins required for the successful mitotic transmission of acentric chromosomes to daughter cells. These studies reveal unsuspected, distinct forces that likely act on all chromosomes during mitosis independent of kinetochore-microtubule attachments.

cell biology↗

Aurora B-mediated exclusion of HP1a from late-segregating chromatin prevents formation of micronuclei

Late-segregating acentric chromosomes pose a serious risk to genomic integrity when they are excluded from dividing daughter nuclei and form damage-prone micronuclei. Insight into the cellular mechanisms that prevent the formation of micronuclei from acentrics come from studies demonstrating that acentrics reincorporate into daughter telophase nuclei by passing through Aurora B kinase-dependent channels in the nuclear envelope of Drosophila neuroblasts. Here, we uncover a mechanism of nuclear envelope channel formation in which localized concentrations of Aurora B preferentially phosphorylate H3(S10) on heterochromatic acentrics and their associated DNA tethers. This phosphorylation event prevents HP1a from associating with heterochromatin and results in localized inhibition of nuclear envelope reassembly on endonuclease- and X-irradiation-induced acentrics and the main daughter nuclei at the sites of acentric entry to promote the formation of channels. Finally, we find that HP1a also specifies initiation sites of nuclear envelope reassembly on undamaged chromatin. Taken together, these results demonstrate that Aurora B-mediated regulation of HP1a-chromatin interactions plays a key role maintaining genome integrity by locally preventing nuclear envelope assembly and facilitating incorporation of late-segregating acentrics into daughter nuclei.

cell biology↗

A comparison of root and shoot hydraulics, aquaporin expression and leaf gas exchange between two grapevine cultivars reveals differences in hydraulic control mediated by aquaporins

Hydraulics of plants that take different strategies of stomatal control under water stress are still relatively poorly understood. Here we explore how root and shoot hydraulics, gas exchange, aquaporin expression and abscisic acid (ABA) concentration in leaf xylem sap ([ABA]xylem) may be involved and coordinated. A comparison in responses to mild water stress and ABA application was made between two cultivars of Vitis vinifera L. previously classified as isohydric (Grenache) and anisohydric (Syrah). Grenache showed stronger adjustments of leaf, plant, and root hydraulic conductances to decreased soil moisture and a steeper correlation of stomatal conductance (gs) to [ABA]xylem than Syrah resulting in greater conservation of soil moisture, but not necessarily more isohydric behaviour. Under well-watered conditions, changes in vapour pressure deficit (VPD) had a strong influence on gs in both cultivars with adjustments of leaf hydraulic conductance. Grenache was more sensitive to decreases in soil water availability compared to Syrah that rather responded to VPD. There were stronger correlations between plant hydraulic parameters and changes in aquaporin gene expression in leaves and roots of Grenache. Overall, the results reinforce the hypothesis that both hydraulic and chemical signals significantly contribute to the differences in water conservation behaviours of the two cultivars.

plant biology↗

Split personality of Aluminum Activated Malate Transporter family proteins: facilitation of both GABA and malate transport

Plant aluminum activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires the presence of a specific amino acid motif in ALMTs that shares similarity with a GABA-binding site in mammalian GABAA receptors. Here, we explore why TaALMT1-activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA]i) in both wheat root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA]i because TaALMT1 facilitates GABA efflux. TaALMT1-expression also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14CGABA uptake into TaALMT1-expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1F213C) prevented both GABA influx and efflux, and uncoupled the relationship between malate efflux and [GABA]i. We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner regulating each others transport, suggestive of a role for ALMTs in communicating metabolic status.

plant biology↗