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Cook, D.

Publications and source records attributed to Cook, D..

At least 19 recordsLinked to original sources

Remodeled cephalic arch geometry promotes disturbed flow at pre-maturation flow rates in hemodialysis patients

In the United States, roughly 550,000 people receive routine hemodialysis for end-stage renal disease. This treatment requires an arteriovenous access, most commonly a brachiocephalic fistula. However, these accesses often fail due to stenosis in the cephalic arch (CA), a common complication whose underlying causes remain unclear (Bennet et al., 2015). To characterize the hemodynamic environment in the CA, we employed patient-specific millifluidic models that were perfused with blood-mimicking fluid containing fluorescently labelled beads to visualize flow behaviors. We perfused our models across physiologic (28-45 mL/min) and elevated (60-423 mL/min) flow rates and quantified wall shear stress (WSS) and streamline angle as a measure of flow disturbance. Our findings show that the bulk curvature and remodeled wall topography each create regions of persistently low WSS, consistent with prior clinical observations (Hammes et al., 2016). Moreover, remodeled wall topography promotes disturbed flow at elevated flow rates, a hemodynamic profile associated with various vascular pathologies (Chiu & Chien, 2011). Independently performed computational fluid dynamics (CFD) modeling complements these results, showing that remodeled wall topography promotes vortex formation at elevated flow rates, as assessed by Q-criterion. Collectively, our experimental and computational results provide strong evidence for geometry-driven disturbed flow in the CA at elevated flow rates. Notably, we observed disturbed flow at flow rates as low as 81 mL/min, far below the 600 mL/min required for hemodialysis. Disturbed flow thus offers a plausible mechanism that relates access flow rates to the vascular pathologies that precede access failure. Significance StatementHemodialysis requires an arteriovenous fistula (AVF) that must remodel and mature to withstand chronically elevated blood flow rates. However, how remodeled vessel geometry interplays with elevated flow to shape local hemodynamics remains poorly understood. Here, we used millifluidic models of the cephalic arch (CA) to show that vessel geometry and elevated flow rates promote regions of low wall shear stress and disturbed flow. Notably, geometry-driven disturbed flow is observed at flow rates above physiologic levels but well below 600 mL/min, the flow rate necessary for adequate dialysis. Because disturbed flow is injurious to the endothelium, our findings are the first to show that vascular damage begins before fistula maturation.

biophysics↗

Evolution of supernumerary chromosomes in wheat blast fungal pathogens

A genome of Pyricularia oryzae (synonym Magnaporthe oryzae), the fungus that causes blast disease on diverse grass species, has seven core chromosomes and may contain supernumerary mini-chromosomes. The P. oryzae Triticum (PoT) pathotype is the phylogenetic lineage responsible for devastating epidemics of wheat blast disease. Genomic analysis of wheat blast field isolates from the initial outbreak in 1985 in Brazil through recent field isolates in South America revealed dynamic presence and structure of mini-chromosomes. Two "earliest" field isolates representing founder lineages for the Triticum pathotype contain similar mini-chromosomes. Another PoT founder isolate from 1986 and 37 out of 39 Triticum field isolates collected between 1986 and 1992 lack mini-chromosomes. Mini-chromosomes present in the founder strains each contain two copies of the PWT7 wheat blast avirulence gene, and PWT7 was lost from subsequent early strains through mini-chromosome loss. Almost all PoT field isolates from 2005 to 2020 have regained mini-chromosomes in which PWT7 sequences have been replaced by other sequences. Telomere-to-telomere assemblies of 11 mini-chromosomes identified two major mini-chromosome types in the South American PoT population, and demonstrated significant within-mini-chromosome sequence alterations as well as recombination with other mini-chromosomes or core chromosome ends. Additionally, our data indicate horizontal mini-chromosome transfer between Pyricularia species, resulting in nearly identical genomic fragments shared between P. oryzae and Pyricularia pennisetigena isolates in the PWT4 avirulence gene region. Our genomic analysis depicts the dynamic mini-chromosome compartment in the diverse South American Triticum field population through time, indicating important roles for mini-chromosomes in pathogen adaptation and pathogenicity.

evolutionary biology↗

Systemic and tumor intrinsic expansion of FCRL5 expressing B cells associates with poor response to Bacillus Calmette-Guerin immunotherapy in patients with non-muscle invasive bladder cancer

The majority of patients treated with Bacillus Calmette-Guerin (BCG) immunotherapy, for non-muscle invasive bladder cancer (NMIBC), experience early recurrence due to pre-existing mucosal immune dysfunction. Since B cell are mucosal immune sentinels, we characterized the systemic and local B cell responses in 45 patients with NMIBC. Expansion of circulating atypical B cells (ABCs) following repeated BCG instillation, expanded IgG autoantibody repertoire, progressive IgG reactivity against BCG antigens, and higher tumor IgG deposition, were features of patients who recurred early. Integrated spatial immunophenotyping and single cell spatial transcriptomic analysis of corresponding tumors revealed increased ABCs within tertiary lymphoid structures, and co-localization with PD-1 B cells, regulatory T cells, and CD163 macrophages. Independent validation in two patient cohorts (total n = 409), revealed a significant association between high expression of the ABC specific, FCRL5, and poor outcomes. Our study identifies ABCs as key mediators of poor response to BCG in patients with high-risk NMIBC.

cancer biology↗

Distinct modes of sequence evolution and epigenetic modifications underpin the origins of Starship-mediated variation in Pyricularia fungal plant pathogens

Fungal pathogens display remarkable variation in genome content and organization that directly impacts their survival and host interactions. Although numerous models have been proposed to explain the origins of this variation, they generally fail to explain or predict the mechanisms that generate the genome variation observed in natural populations. Starships are a recently discovered group of giant fungal transposons that carry dozens of genes as cargo and horizontally transfer both within and between species. Here, we identify the features of a newly defined "Starship compartment" in the major fungal plant pathogen Pyricularia oryzae. We test the hypothesis that the Starship compartment makes distinct contributions to fungal genome evolution by explicitly comparing its transferability, mutability, and epigenetic modifications with those of the canonical core and accessory compartments. To enable this, we developed an updated and user-friendly version of the annotation tool stargraph for the comprehensive annotation of Starships and Starship-like regions. Using this approach, we identified two distinct families of Starships and related Starship-like regions in P. oryzae that differ in their activity, impacts on genome organization, modes of sequence evolution, and epigenetic modifications. Elements from the more active family exhibit higher rates of structural variation than all other genomic compartments in the predominantly clonal isolates infecting rice. Both families of Starships encode specific suites of known effector sequences that contribute to plant disease and Starship activity accounts for avirulence gene turnover, which suggests that evolutionary change within the Starship compartment may subsequently impact the evolution of plant-fungal interactions. Starships from the more active family have repeatedly transferred across the Pyricularia genus and tend to be depleted in heterochromatic histone modifications and repeat-induced point mutations. However, contrasting histone modification profiles in this family suggests a genomic conflict between silencing or maintaining Starship activity. Our findings demonstrate that variation in the mode of sequence diversification and epigenetic modification within the Starship compartment underpins the impacts of these giant transposons on fungal genome evolution. We argue for the explicit consideration of not only the Starship compartment but of element-specific dynamics when investigating the evolution of host-fungal interactions.

genomics↗

Capture, Confine, Characterize: High-Throughput Dielectrophoresis-Based Single-Cell Microfluidics Platform to Analyze Mammalian and Yeast Cells Using Raman Spectroscopy

Single-cell analysis technologies are pivotal in unraveling complex biological mechanisms, yet existing platforms are often limited to sequencing-based end-point measurements, which fail to capture live cell dynamics. Here, we present a microfluidic- microelectronic device, the Microfluidic dielectrophoretic Arresting System (MiDAS) that employs dielectrophoresis (DEP) for high-throughput single-cell and droplet trapping in a compact array. We tested multiple trap geometries, including a 20 m-diameter DEP trap for polymer microbeads, fungal and mammalian cells, and a 40 m-diameter trap for water-in-oil droplets. The platform demonstrates broad sample compatibility, reliably immobilizing cells and beads of varying sizes. By integrating optical imaging and Raman spectroscopy, we enable rapid, non-destructive interrogation of individual cells with temporal resolution. We describe different modes of MiDAS operation to trap and manipulate single-cells or reverse emulsion droplets on demand, with applications in droplet microfluidics. Our MiDAS platforms simple fabrication, robust performance, and broad compatibility with diverse sample types position it as a versatile tool with transformative potential for single-cell analysis, offering researchers an innovative approach to interrogate cellular dynamics with unprecedented precision and throughput.

bioengineering↗

An increased excitation and inhibition onto CA1 pyramidal cells sets the path to Alzheimer s disease

Synapses are critical targets of Alzheimers disease (AD), a highly prevalent neurodegenerative disease associated with accumulation of extracellular amyloid-{beta} peptides. Although amyloidosis and aggregation of the 42-amino acid amyloid-{beta} (A{beta}42) have long been considered pathogenic triggers for AD, clinical evidence linking high levels of A{beta}42 with normal cognition challenges this hypothesis. To resolve this conundrum on the role of A{beta}42 in regulating synaptic activity, we used an adeno-associated viral vector approach that triggers extracellular accumulation of A{beta}42 and spatial memory impairment. We show that A{beta}42 leads to an early increase in excitatory and proximal inhibitory synaptic transmission onto hippocampal CA1 pyramidal cells, and an increased expression of the glutamate transporter GLT-1 in these cells. A{beta}42 accumulation does not cause early cognitive deficits unless accompanied by an increased neuronal GLT-1 expression, suggesting this transporter is a critical mediator of A{beta}42s effects. These findings unveil key molecular and cellular mechanisms implicated with AD pathogenesis.

neuroscience↗

DNA data (genome skims and metabarcodes) paired with chemical data demonstrate utility for retrospective analysis of forage linked to fatal poisoning of cattle

Prepared and stored feeds, fodder, silage, and hay may be contaminated by toxic plants resulting in the loss of livestock. Several poisonous plants have played significant roles in livestock deaths from forage consumption in recent years in the Western United States including Salvia reflexa. Metagenomic data, genome skims and metabarcodes, have been used for identification and characterization of plants in complex matrices including diet composition of animals, mixed forages, and herbal products. Here, chemistry, genome skims, and metabarcoding were used to retrospectively describe the composition of contaminated alfalfa hay from a case of Salvia reflexa poisoning that killed 165 cattle. Genome skims and metabarcoding provided similar estimates of the relative abundance of the Salvia in the hay samples when compared to chemical methods. Additionally, genome skims and metabarcoding provided similar estimates of species composition in the contaminated hay and rumen contents of poisoned animals. The data demonstrate that genome skims and DNA metabarcoding may provide useful tools for plant poisoning investigations.

genomics↗

LAG3 marks activated but hyporesponsive NK cells.

Natural Killer (NK) cells are critical for immunosurveillance yet become dysfunctional in contexts such as chronic stimulation by viral infections or cancer. This phenomenon is similar to T cell exhaustion but less well characterized, which limits therapeutic interventions. As shown for T cells, NK cells often display an increased expression of immune checkpoint proteins (ICP) following chronic stimulation, and ICP blockade therapies are currently being explored for several cancer types, which have remarkable patient benefits. Nevertheless, the nature of ICP expression in NK cells is still poorly documented. In this study, we aimed to identify the conditions that lead to and the phenotype of immune checkpoint LAG3 (Lymphocyte-activation gene 3) expressing NK cells. Using various experimental models, we found that LAG3 is expressed by murine NK cells upon activation in different contexts, including in response to cancer and acute viral infections. LAG3 marks a subset of immature, proliferating and activated cells, which, despite activation, have a reduced capacity to respond to a broad range of stimuli. Further characterization also revealed that LAG3+ NK cells exhibit a transcriptional signature similar to that of exhausted CD8+ T cells. Taken together, our results support the use of LAG3 as a marker of dysfunctional NK cells across diverse chronic and acute inflammatory conditions.

immunology↗

Highly dynamic supernumerary mini-chromosomes in a Magnaporthe oryzae strain

Magnaporthe oryzae (syn. Pyricularia oryzae), the causative agent of devastating crop diseases, exhibits remarkable genomic plasticity that contributes to its adaptability and pathogenicity. Individual M. oryzae strains may contain supernumerary mini-chromosomes, which are dispensable and highly repetitive. Here, we explored the stability of two mini-chromosomes of a Lolium strain isolated in the US, TF05-1, in which one mini-chromosome contains sequences nearly identical to the mini-chromosome of the wheat isolate B71 from Bolivia. The discordance of their phylogenetic relationships based on genomic polymorphisms in core chromosomes and polymorphisms in mini-chromosomes indicated horizontal transfer of the mini-chromosome. Karyotyping analysis and genome sequencing analysis found variation in numbers and sizes of mini-chromosomes among asexual monoconidial progeny of TF05-1. Rearrangement within mini-chromosomes occurred frequently in the TF05-1 progeny. We characterized an intrachromosomal rearrangement presumably mediated by a palindrome repeat. The rearrangement resulted in a 300-kb deletion and a 900-kb duplication. The susceptibility to structural variation in mini-chromosomes may be associated with repetitive features and the high activity of transposable elements in mini-chromosomes, in which many intact retrotransposons were recently inserted, largely unmethylated, and likely have yet to be silenced by fungal genome defense mechanisms such as repeat-induced point mutation.

genomics↗

Chromatin Remodelling in Damaged Intestinal Crypts Orchestrates Redundant TGFβ and Hippo Signalling to Drive Regeneration

Cell state dynamics underlying regeneration are under-characterized. Intestinal damage prompts reprogramming into revival stem cells (revSCs) that reconstitute Lgr5+ intestinal stem cells (ISCs). Single nuclei multiomics of chromatin accessibility and transcriptomes during regeneration from irradition showed revSCs display epigenetic profiles shared with ISCs and differentiated lineages. Furthermore, while revSC genes are accessible throughout homeostatic epithelia, damage-induced global alterations in crypt and revSC chromatin converge on TGF{beta}, as well as Hippo pathways. We show TGF{beta} directly induces functional revSCs and demonstrate individual revSCs form organoids with reconstituted Lgr5+ ISCs. Despite this, loss of TGF{beta} signalling yielded mild regenerative defects. In contrast, interference in both Hippo and TGF{beta} abolished revSCs, precluded generation of new ISCs and led to rapid intestinal collapse. Thus, the epithelium is poised to engage the revSC regenerative program that relies on crypt-localized, transient morphogen cues that function in a compensatory manner to support intestinal regeneration.

molecular biology↗

Mitotic Activity and DNA Maintenance of Adult Neural Stem Cells is Regulated by Beclin1

Beclin1 is a tumor suppressor gene that can regulate proliferation under pathological conditions. Whether Beclin1 has a role in regulating proliferation in physiological conditions remains unknown. Here, through the creation of an inducible transgenic mouse that removes Beclin1 from adult neural stem and progenitor cells (NSPCs) and their progeny we uncovered that Beclin1 is required cell-autonomously to sustain proliferating NSCPs in vivo and ex vivo. Flow cytometry analysis and single-cell RNA-sequencing show that Beclin1 is required for mitosis. Additional analysis of the proliferating NSPCs resolved by stage of cell cycle uncovered that Beclin1-null cells have a distinct differential developmental trajectory accompanied by downregulation of genes involved in chromosomal maintenance and upregulation of cell stress genes upon cell cycle exit. These effects align with DNA damage in Beclin1-null cells and ultimately result in less adult-born granular neurons. Together these data identify Beclin1 as a novel regulator of mitosis in adult NSPCs.

neuroscience↗

Phase-changing citrate macromolecule combats oxidative pancreatic islet damage, enables islet engraftment and function in the omentum

Clinical outcomes for total-pancreatectomy followed by intraportal islet autotransplantation (TP-IAT) to treat chronic pancreatitis (CP) patients are suboptimal due to the inflammatory state of the patients pancreas, oxidative tissue damage during the isolation process, and the harsh engraftment conditions in the livers vasculature, which include ischemia-reperfusion injury, and instant blood-mediated inflammatory reactions. We describe the use of the thermoresponsive, antioxidant macromolecule poly(polyethylene glycol citrate-co-N-isopropylacrylamide) (PPCN) to protect islet redox status and function in vitro and in vivo and to create a viable extrahepatic islet engraftment site in the abdomen. PPCN in aqueous media transitions from a liquid to an elastic hydrogel when exposed to body temperature via temperature-induced macromolecular self-assembly. Islets entrapped in the PPCN hydrogel and exposed to oxidative stress remain functional and support long-term euglycemia, in contrast to islets entrapped in a biologic scaffold (BS). When applied to the omentum of non-human primates (NHPs), PPCN is well-tolerated, safe, and mostly resorbed without fibrosis at 3 months post-implantation. To obtain autologous islets, a partial pancreatectomy was performed, followed by STZ administration to induce diabetes and destroy any remaining endogenous islets. Application of the autologous islets to the momentum using PPCN restored normoglycemia with minimal insulin requirements for over 100 days. These results support the use of PPCN as a scaffold for minimally invasive delivery of islets to the omentum of pancreatitis patients and highlight the importance of scaffold antioxidant properties as a new mechanism to protect islet function and maximize long-term autologous graft performance. One Sentence SummaryOmentum islet transplantation using a thermoresponsive, antioxidative polymer supports autologous islet viability and function in nonhuman primates.

bioengineering↗

Using recurrent neural networks to detect supernumerary chromosomes in fungal strains causing blast diseases

The genomes of the fungus Magnaporthe oryzae that causes blast diseases on diverse grass species, including major crop plants, have indispensable core-chromosomes and may contain one or more additional supernumerary chromosomes, also known as mini-chromosomes. The mini-chromosome is speculated to play a role in fungal biology, provide effector gene mobility, and may transfer between strains. To understand and study the biological function of mini-chromosomes, it is crucial to be able to identify whether a given strain of M. oryzae possesses a mini-chromosome. In this study, we applied recurrent neural network models, more specifically, Bidirectional Long Short-Term Models (Bi-LSTM), for classifying DNA sequences as core-or mini-chromosomes. The models were trained with sequences from multiple available core- and mini-chromosome assemblies. The trained model was then used to predict the presence of the mini-chromosome in a global collection of M. oryzae isolates using short-read DNA sequences. The model predicted that the mini-chromosome was prevalent in M. oryzae isolates, including those isolated from rice, wheat, Lolium and many other grass species. Interestingly, 23 recent wheat strains collected since 2005 all carried the mini-chromosome, but none of nine early strains collected before 1991 had the mini-chromosome, indicating the preferential selection for strains carrying the mini-chromosome in recent years. Based on the limited sample size, we found the presence of the mini-chromosome in isolates of pathotype Eleusine was not as high as isolates of other pathotypes. The deep learning model was also used to identify assembled sequence contigs that were derived from the mini-chromosome and partial regions on core-chromosomes potentially translocated from a mini-chromosome. In summary, our study has developed a reliable method for categorizing DNA sequences and showcases an application of recurrent neural networks in the field of predictive genomics.

genomics↗

Natural genomic variation in rice blast genomes is associated with specific heterochromatinmodifications

Genome organization in eukaryotes exhibits non-random patterns tied to transcription, replication, and chromatin. However, the driving forces across these processes, and their impacts on genome evolution remain unclear. To address this, we analyzed sequence data from 86 Magnaporthe oryzae isolates, a globally important plant pathogenic fungus, alongside new reference genomes, to investigate DNA sequence variation and the epigenome. Histone modification profiles were obtained through genome-wide chromatin immunoprecipitation-sequencing of the four new reference strains, which revealed that repressive histone marks (H3K27me3, H3K9me3) were associated with SNP and INDEL frequency. Densely grouped SNPs were found to reside in heterochromatin, often outside transposable elements, highlighting the link between heterochromatin and DNA variation. Even when controlling for selection, silent SNP frequency/kb was higher in H3K27me3-associated genes. Effector genes, key to pathogenicity, also displayed this trend. Comparing the reference strains, euchromatic regions were often syntenic, while heterochromatic regions trended towards non-syntenic. Heterochromatin emerged as a major factor associated with diverse DNA variations in M. oryzae populations, even when accounting for selective pressure. This underscores heterochromatins pivotal role in shaping genetic diversity in these mainly asexually reproducing fungi.

evolutionary biology↗

Three-dimensional chromatin organization promotes genome evolution in a fungal plant pathogen

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly structured in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus.

microbiology↗

Best: A Tool for Characterizing Sequencing Errors

SummaryPlatform-dependent sequencing errors must be understood to develop accurate sequencing technologies. We propose a new tool, best (Bam Error Stats Tool), for efficiently quantifying and summarizing error types in sequenced reads. best ingests reads aligned to a high-quality reference assembly and produces per-read metrics, summary statistics, and stratified metrics across genomic intervals. We show that best is 16 times faster than a prior method. In addition to being useful to support development that improves the accuracy of sequencing platforms, best can also be applied to evaluate and improve other experimental factors such as library preparation and error correction methods. Availability and implementationbest is an open-source command-line utility available on Github (github.com/google/best) under an MIT license. Contactdanielecook@google.com

bioinformatics↗

Computational Speed-Up of Large-Scale, Single-Cell Model Simulations Via a Fully-Integrated SBML-Based Format

SummaryLarge-scale and whole-cell modeling has multiple challenges, including scalable model building and module communication bottlenecks (e.g. between metabolism, gene expression, signaling, etc). We previously developed an open-source, scalable format for a large-scale mechanistic model of proliferation and death signaling dynamics, but communication bottlenecks between gene expression and protein biochemistry modules remained. Here, we developed two solutions to communication bottlenecks that speed up simulation by ~4-fold for hybrid stochastic-deterministic simulations and by over 100-fold for fully deterministic simulations. Availability and ImplementationSource code is freely available at https://github.com/birtwistlelab/SPARCED/releases/tag/v1.1.0 implemented in python, and supported on Linux, Windows, and MacOS (via Docker). ContactMarc Birtwistle mbirtwi@clemson.edu Supplementary informationN/A

systems biology↗

More than a ligand: PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I interferon pathway.

Targeting the PD-1/PD-L1 axis has transformed the field of immune-oncology. While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced infection with oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 suppressed type I interferon by promoting a metabolic shift characterized by enhanced glucose uptake and glycolysis rate. Lactate generated from glycolysis was the key metabolite responsible for inhibiting type I interferon responses and enhancing oncolytic virus infection in PD-L1-expressing cells. In addition to adding mechanistic insight into PD-L1 intrinsic function and showing that PD-L1 has a broader impact on immunity and cancer biology besides acting as a ligand for PD-1, our results will also help guide the numerous efforts currently ongoing to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials. Once sentence summaryPD-L1 promotes oncolytic virus efficacy.

cancer biology↗