Biology subjects

Craig, J.

Publications and source records attributed to Craig, J..

13 recordsLinked to original sources

bioRxiv2026

Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)

Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and crystal structures of TvPPi-PFK are reported. TvPPi-PFK is the first reported eukaryotic PPi-PFK structure. TvPPi-PFK retains the overall PPi-PFK topology observed in bacterial PPi-PFK including conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. In addition to the catalytic PPi-PFK binding sites, TvPPi-PFK has two additional ligand binding sites. The first binds AMP usurped during protein production and helps stabilize the TvPPi-PFK tetramer. A second ligand binding site was observed in proximity to the AMP-binding site and accommodates sugar phosphates soaked into preformed crystals. This sugar phosphates binding site is distinct from the TvPPi-PFK active site that binds fructose-6-phosphate. Future mutagenesis and activity studies are planned to determine the relevance of both sites. SynopsisThe production, crystallization, and crystal structures of a pyrophosphate-dependent phosphofructokinase from Trichomonas vaginalis (TvPPi-PFK) are reported. TvPPi-PFK has a prototypical PPi-PFK active site as well as unexpected AMP and sugar-phosphate binding sites at the dimer interface.

biochemistry↗

bioRxiv2025

An integrated DNA interactome and transcriptome profiling reveals a PU.1/enhancer RNA-mediated Feed-forward Regulatory Loop Regulating monocyte/macrophage development and innate immune functions

High expression of the myeloid master ETS transcription factor PU.1 drives the development of monocyte/macrophage (Mono/M{Phi}), a crucial cellular component of the innate immune system. Disruptions in normal expression patterns of PU.1 are linked to a variety myeloid malignancy and immune diseases. It is evidenced that PU.1 binds to and modulates enhancers of several myeloid genes. While noncoding RNAs transcribed from noncoding genes at the enhancers are increasingly reported to be involved in enhancer regulation, the crosstalk between PU.1 and noncoding RNAs in enhancer-mediated myeloid gene regulation in Mono/M{Phi} differentiation and immune response has not been systematically investigated. In this study, we interrogated the PU.1-mediated transcriptome and cistrome with our comprehensive collection of putative and verified enhancers. Among a repertoire of noncoding genes present at PU.1-bound enhancers, we discovered that PU.1 acts as a potent transcription factor inducer of the noncoding RNA LOUP, which we previously identified as an RNA inducer of PU.1. The genomic region within the LOUP locus occupied by PU.1 is characterized by the epigenetic features of a myeloid-specific super-enhancer. Targeted disruption of the PU.1-binding motifs resulted in the downregulation of LOUP promoter activity. Depletion of LOUP reduced the expression of Mono/M{Phi} cell markers as well as the transcriptional program associated with Mono/M{Phi} differentiation Mono/M{Phi} innate defense mechanisms, including phagocytosis, antimicrobial activity, and chemoattractant cytokine production. LOUP induces Mono/M{Phi} phagocytic activities. Collectively, our findings indicate that PU.1 and enhancer RNA LOUP are biomolecular components of an unidentified feed-forward loop that promotes their mutual expression, contributing to Mono/M{Phi} differentiation and innate immune functions. The identification of the PU.1/LOUP regulatory circuit provides valuable insights into the mechanisms underlying cell-type and gene-specific enhancer activity and Mono/M{Phi} biology, as well as significant implications for advancing our understanding of immune diseases and myeloid malignancies.

molecular biology↗

bioRxiv2025

Advanced Manufacturing of Coil-Reinforced Multilayer Vascular Grafts to Optimize Biomechanical Performance

Small diameter vascular grafts require a complex balance of biomechanical properties to achieve target burst pressure, arterial compliance-matching, and kink resistance to prevent failure. Iterative design of our multilayer vascular was previously used to achieve high compliance while retaining the requisite burst pressure and suture retention strength for clinical use. To impart kink resistance, a custom 3D solution printer was used to add a polymeric coil to the electrospun polyurethane graft to support the graft during bending. The addition of this reinforcing coil increased kink resistance but reduced compliance. A matrix of grafts were fabricated and tested to establish key structure-property relationships between coil parameters (spacing, diameter, modulus) and biomechanical properties (compliance, kink radius). A successful graft design was identified with a compliance similar to saphenous vein grafts (4.1 {+/-} 0.4 %/mmHgx10-2) while maintaining comparable kink resistance to grafts used currently in the clinic. To explore graft combinations that could increase graft compliance to match arterial values while retaining this kink resistance, we utilized finite element (FE) models of compliance and kink radius that simulated experimental testing. The FE-predicted graft compliance agreed well with experimental values. Although the kink model over-predicted the experimental kink radius values, key trends between graft parameters and kink resistance were reproduced. As an initial proof-of-concept, the validated models were then utilized to parse through a targeted graft design space. Although this initial parameter range tested did not yield a graft that improved upon the previous balance of graft properties, this combination of advanced manufacturing and computational framework paves the way for future model-driven design to further optimize graft performance. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/633374v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@faca56org.highwire.dtl.DTLVardef@f9dccforg.highwire.dtl.DTLVardef@fbb5corg.highwire.dtl.DTLVardef@d5c021_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Lower perceived stress enhances neural synchrony in perceptual and attentional cortices during naturalistic processing

Perceived stress is the subjective appraisal of the level of stress experienced by an individual in response to external or internal demands. Recent research on perceived stress has highlighted its role in influencing cognition, leading to a disruption in cognitive processes, such as emotional processing, attention, and perception. However, most neuroimaging studies examining stress have used static stimuli (e.g., still images) that do not encapsulate real-life multimodal processing in the brain. The current research uses data from the Naturalistic Neuroimaging Database (v2.0; Aliko et al., 2020) to examine differences in neural synchrony (as measured by intersubject correlations; ISCs) associated with perceived stress using functional magnetic resonance imaging (fMRI). We evaluated how self-reported perceived stress levels influence neural synchrony patterns in response to different naturalistic stimuli by examining the differences in neural synchrony between individuals with low and high perceived stress levels. We determined that lower perceived stress was observed with greater neural synchrony areas associated with perceptual and attention processing, including the lateral occipital cortex, superior temporal gyrus, superior parietal lobule, orbital frontal cortex, and the occipital pole. These results indicate that high levels of perceived stress heavily alter neural processing of complex audiovisual stimuli. Together, these results provide evidence that perceived stress influences cognitive processing in everyday life.

neuroscience↗

bioRxiv2023

Psychological well-being modulates neural synchrony during naturalistic fMRI

Psychological well-being (PWB) is a combination of self-acceptance, life purpose, personal growth, positive relationships, and autonomy, and has a significant relationship with physical and mental health. Previous studies using resting-state functional magnetic resonance imaging (fMRI) and static picture stimuli have implicated the anterior cingulate cortex (ACC), posterior cingulate cortex (PCC), orbitofrontal cortex (OFC), insula and thalamus in PWB, however, the replication of associations across studies is scarce, both in strength and direction, resulting in the absence of a model of how PWB impacts neurological processing. Naturalistic stimuli better encapsulate everyday experiences and can elicit more "true-to-life" neurological responses, and therefore may be a more appropriate tool to study PWB. The current study seeks to identify how differing levels of PWB modulate neural synchrony in response to an audiovisual film. With consideration of the inherent variability of the literature, we aim to ascertain the validity of the regions previously mentioned and their association with PWB. We identified that higher levels of PWB were associated with heightened neural synchrony in the bilateral OFC and left PCC, and that lower levels of PWB were associated with heightened neural synchrony in the right temporal parietal junction (TPJ) and left superior parietal lobule (SPL), regions related to narrative processing. Taken together, this research confirms the validity of several regions in association with PWB and suggests that varying levels of PWB produce differences in the processing of a narrative during complex audiovisual processing.

neuroscience↗

bioRxiv2023

Trait level somatic arousal modulates fMRI neural synchrony to naturalistic stimuli

Somatic arousal refers to the physiological and bodily responses that occur in reaction to different emotional and psychological stimuli and is a crucial component of the fight or flight response. Symptoms associated with higher levels of somatic arousal such as higher heart and respiration rates have been shown to impact the blood oxygenation level dependent (BOLD) signal during functional magnetic resonance imaging (fMRI) studies. Differences in baseline levels of somatic arousal may therefore modulate the brains response to incoming stimuli during fMRI. Previous studies typically investigate somatic arousal as a state, rather than as a trait, in which some individuals are more likely to have heightened physiological responses to psychological stimuli, causing the neurological mechanisms behind baseline somatic arousal levels to remain poorly understood. The current study seeks to identify how differing levels of baseline somatic arousal modulate neural synchrony in response to an audiovisual film. We hypothesize that individuals with higher levels of somatic arousal will show overall heightened neural synchrony in response to a complex audiovisual stimulus. We identified that higher levels of somatic arousal are associated with widespread neural synchrony across the brain, including frontal gyri, parietal and temporo-occipital cortices. Taken together, this research suggests that baseline somatic arousal levels should be measured during naturalistic fMRI paradigms, as baseline somatic arousal levels may have a profound influence on synchronous neural activity.

neuroscience↗

bioRxiv2023

Mapping the Changing Neural Architecture of Narrative Processing Using Naturalistic Stimuli: an fMRI Study

A narrative is a coherent representation of actual or fictional events designed to connect experiences. Narratives provide a unique opportunity to investigate brain functions in scenarios more closely resembling real-world experiences. However, most neuroimaging studies examining narrative formation have utilized static stimuli that fail to capture the intricacies of narrative construction in everyday life, particularly how cognitive demands change over the course of narrative processing. The current research uses functional magnetic resonance imaging (fMRI) to examine dynamic narrative processing over the course of a full-length audiovisual narrative. We examined changes in neural synchrony (as quantified by intersubject correlations) in areas related to semantic memory, episodic memory, and visuospatial attention between the beginning, middle, and end of the narrative. Results from two experiments identified two core narrative processing networks responsible for constructing coherent representations across extended timescales. The first network is associated with the early narrative construction, and includes the right intraparietal sulcus/superior parietal lobule, bilateral angular gyrus, bilateral precuneus, and left fusiform gyrus. The second network consists of the right ventral frontal cortex and bilateral parahippocampal cortices, and is associated with longer term narrative integration. Together, these regions provide the framework for successful narrative processing during naturalistic stimuli.

neuroscience↗

bioRxiv2023

High throughput functional profiling of genes at intraocular pressure loci reveals distinct networks for glaucoma

INTRODUCTIONPrimary open angle glaucoma (POAG) is a leading cause of blindness globally. Characterised by progressive retinal ganglion cell degeneration, the precise pathogenesis remains unknown. Genome-wide association studies (GWAS) have uncovered many genetic variants associated with elevated intraocular pressure (IOP), one of the key risk factors for POAG. This study sought to investigate the morphological and transcriptional consequences of perturbation of key genes at IOP loci in trabecular meshwork cell (TMC); the cellular regulators of IOP. We aimed to identify genetic and morphological variation that can be attributed to TMC dysfunction and raised IOP in POAG. METHODS62 genes across 55 loci were knocked-out in a primary human TMC line. Each knockout group, including five non-targeting control groups, underwent single-cell RNA-sequencing (scRNA-seq) for differentially-expressed gene (DEG) analysis. Multiplexed fluorescent staining of key organelles, was coupled with high-throughput microscopy for single-cell morphological analysis using CellProfiler image analysis. RESULTSAcross many of the individual gene knockouts scRNA-seq highlighted genes relating to matrix metalloproteinases and interferon-induced proteins. Our work has prioritised genes at four loci of interest to identify gene knockouts that may contribute to the pathogenesis of POAG, including ANGPTL2, LMX1B, CAV1, and KREMEN1. Three genetic networks of gene knockouts with similar transcriptomic profiles were identified (ABO / CAV1 / MYOC, ANGPT2 / PKHD1 / TNS1 / TXNRD2, and CAPZA1 / KALRN / LMO7 / PLEKHA7 / GNB1L / TEX41), suggesting a synergistic function in trabecular meshwork cell physiology. TEK knockout caused significant upregulation of nuclear granularity on morphological analysis, whilst knockout of TRIOBP, TMCO1 and PLEKHA7 increased granularity and intensity of actin and the cell-membrane. CONCLUSIONHigh throughput analysis of cellular structure and function through multiplex fluorescent single-cell analysis and scRNA-seq assays enabled the direct study of genetic perturbations at the single-cell resolution. This work provides a framework for investigating the role of genes in the pathogenesis of glaucoma and heterogenous diseases with a strong genetic basis.

genetics↗

bioRxiv2023

Deep Learning-based Identification of Intraocular Pressure-Associated Genes Influencing Trabecular Meshwork Cell and Organelle Morphology

PURPOSEThe exact pathogenesis of primary open-angle glaucoma (POAG) is poorly understood. Genome-wide association studies (GWAS) have recently uncovered many loci associated with variation in intraocular pressure (IOP); a crucial risk factor for POAG. Artificial intelligence (AI) can be used to interrogate the effect of specific genetic knockouts on the morphology of trabecular meshwork cells (TMCs), the regulatory cells of IOP. METHODSSixty-two genes at fifty-five loci associated with IOP variation were knocked out in primary TMC lines. All cells underwent high-throughput microscopy imaging after being stained with a five-channel fluorescent cell staining protocol. A convolutional neural network (CNN) was trained to distinguish between gene knockout and normal control cell images. The area under the receiver operator curve (AUC) metric was used to quantify morphological variation in gene knockouts to identify potential pathological perturbations. RESULTSCells where RALGPS1 had been perturbed demonstrated the greatest morphological variation from normal TMCs (AUC 0.851, SD 0.030), followed by LTBP2 (AUC 0.846, SD 0.029) and BCAS3 (AUC 0.845, SD 0.020). Of seven multi-gene loci, five had statistically significant differences in AUC (p<0.05) between genes, allowing for pathological gene prioritisation. The mitochondrial channel most frequently showed the greatest degree of morphological variation (33.9% of cell lines). CONCLUSIONSWe demonstrate a robust method for functionally interrogating genome-wide association signals using high-throughput microscopy and AI. Genetic variations inducing marked morphological variation can be readily identified, allowing for the gene-based dissection of loci associated with complex traits.

genetics↗

bioRxiv2020

Naegleria fowleri: protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brains frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogens high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. N. fowleri genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

molecular biology↗

bioRxiv2020

Loss of the Pioneer Factor FOXA1 Results in Genome-wide Epigenetic Reprogramming and activation of Interferon-Response Genes including CD274/PD-L1

Forkhead Box A1 (FOXA1) is a pioneer transcription factor critical in epigenetic regulation of chromatin and cell fate determination. Reduced FOXA1 expression is an independent predictor of poor overall survival in bladder cancer patients. However, the impact of FOXA1 loss on chromatin epigenetics in bladder cancer is unknown. Therefore, we determined the impact of FOXA1 knock out (KO) on epigenetic modification of chromatin and associated gene expression. We identified 8,230 differentially expressed genes following FOXA1 KO. Surprisingly, Gene Set Enrichment Analysis (GSEA) identified IFN[a]/{gamma} gene expression signatures as enriched following FOXA1 KO. FOXA1 KO induced both increased and decreased numbers of histone 3 lysine 27 acetylation (H3K27ac) sites throughout the genome. As expected, the majority of differences in H3K27ac across genomic areas in FOXA1 KO cells is mapped to intergenic and intronic regions where enhancers reside. In addition, a subset of differential H3K27ac levels were also mapped to proximal promoters and within gene bodies. Integrated analysis of RNA/ChIP-seq data shows changes in gene expression that are mirrored by differences in H3K27ac. Motif analysis of DNA sequence enriched for H3K27ac identified significant increases in transcription factor binding motifs including the interferon sensitive response element (ISRE) and interferon response factors such as IRF1. Moreover, we identified increased H3K27ac of regulatory elements as being associated with several upregulated interferon sensitive genes (ISGs) in FOXA1 KO cells, including CD274/PD-L1. Western blotting and Q-RT-PCR confirmed upregulation of CD274/PD-L1 following FOXA1 KO. Analysis of TCGA data confirmed an inverse relationship between FOXA1 and CD274 in bladder cancer, as well as in other cancers. In summary, we provide evidence of widespread epigenetic reprogramming after FOXA1 KO in bladder cancer cells. Additionally, we provide evidence that FOXA1 KO-induced epigenetic changes contribute to activation of a global interferon-dominant expression signature, including the immune checkpoint target CD274/PD-L1 in a cancer cell-intrinsic manner.

cancer biology↗

bioRxiv2020

The transcriptome of Balamuthia mandrillaris trophozoites for structure-based drug design

Balamuthia mandrillaris, a pathogenic free-living amoeba (FLA), causes cutaneous skin lesions as well as the brain-eating disease: Balamuthia granulomatous amoebic encephalitis (GAE). These diseases, and diseases caused by other pathogenic FLA, Naegleria fowleri or Acanthamoeba species, are minimally studied from a drug discovery perspective; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Chemotherapies for CNS disease caused by B. mandrillaris require vast improvement. Current therapeutics are limited to a small number of drugs that were previously discovered in the last century through in vitro testing or identified after use in the small pool of surviving reports. Using our recently published methodology to identify potentially useful therapeutics, we screened a collection of 85 compounds that have previously been reported to have antiparasitic activity. We identified 59 compounds that impacted growth at concentrations below 220 M. Since there is no fully annotated genome or proteome, we used RNA-Seq to reconstruct the transcriptome of B. mandrillaris and locate the coding sequences of the specific genes potentially targeted by the compounds identified to inhibit trophozoite growth. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing.

genomics↗

bioRxiv2018

Congenital glaucoma with anterior segment dysgenesis in individuals with biallelic CPAMD8 variants

PurposeCongenital glaucoma is a significant cause of irreversible blindness. In some instances glaucoma is associated with developmental abnormalities of the ocular anterior segment, which can impair drainage of aqueous humor, leading to an increase in intraocular pressure.\n\nMethodsGenome sequencing was performed on a parent-proband congenital glaucoma trio, with exome sequencing of 79 additional individuals with suspected primary congenital glaucoma.\n\nResultsWe describe a unique ocular anterior segment dysgenesis associated with congenital glaucoma in four individuals from three unrelated families. In each case, disease was associated with compound heterozygous variants in CPAMD8, a gene of unknown function recently associated with ocular anterior segment dysgenesis, myopia, and ectopia lentis. CPAMD8 expression was highest in neural crest-derived tissues of the adult anterior segment, suggesting that CPAMD8 variation may cause malformation of key drainage structures and the development of high intraocular pressure and glaucoma.\n\nConclusionsThis study reveals a unique genetic cause of childhood glaucoma, and expands the phenotypic spectrum of CPAMD8-associated ocular disease.

genetics↗