Boolean model of growth signaling, cell cycle and apoptosis predicts the molecular mechanism of aberrant cell cycle progression driven by hyperactive PI3K.

The PI3K/AKT signaling pathway plays a role in most cellular functions linked to cancer progression, including cell growth, proliferation, cell survival, tissue invasion and angiogenesis. It is generally recognized that hyperactive PI3K/AKT1 are oncogenic due to their boost to cell survival, cell cycle entry and growth-promoting metabolism. That said, the dynamics of PI3K and AKT1 during cell cycle progression are highly nonlinear. In addition to negative feedback that curtails their activity, protein expression of PI3K subunits has been shown to oscillate in dividing cells. The low-PI3K/low-AKT1 phase of these oscillations is required for cytokinesis, indicating that oncogenic PI3K may directly contribute to genome duplication. To explore this, we construct a Boolean model of growth factor signaling that can reproduce PI3K oscillations and link them to cell cycle progression and apoptosis. The resulting modular model reproduces hyperactive PI3K-driven cytokinesis failure and genome duplication and predicts the molecular drivers responsible for these failures by linking hyperactive PI3K to mis-regulation of Polo-like kinase 1 (Plk1) expression late in G2. To do this, our model captures the role of Plk1 in cell cycle progression and accurately reproduces multiple effects of its loss: G2 arrest, mitotic catastrophe, chromosome mis-segregation / aneuploidy due to premature anaphase, and cytokinesis failure leading to genome duplication, depending on the timing of Plk1 inhibition along the cell cycle. Finally, we offer testable predictions on the molecular drivers of PI3K oscillations, the timing of these oscillations with respect to division, and the role of altered Plk1 and FoxO activity in genome-level defects caused by hyperactive PI3K. Our model is an important starting point for the predictive modeling of cell fate decisions that include AKT1-driven senescence, as well as the non-intuitive effects of drugs that interfere with mitosis.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation provides a crucial epigenetic mark linking genetic variations to environmental influence. We have analyzed array-based DNA methylation profiles of 160 human retinas with co-measured RNA-seq and >8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 methylation quantitative trait loci and 12,505 expression quantitative trait loci) and 13,747 DNA methylation loci affecting gene expression, with over one-third specific to the retina. Methylation and expression quantitative trait loci show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration. Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of macular degeneration pathology by genotype-environment interaction in retina.

Integrating genetic regulation and single-cell expression with GWAS prioritizes causal genes and cell types for glaucoma.

Primary open-angle glaucoma (POAG), characterized by retinal ganglion cell death, is a leading cause of irreversible blindness worldwide. However, its molecular and cellular causes are not well understood. Elevated intraocular pressure (IOP) is a major risk factor, but many patients have normal IOP. Colocalization and Mendelian randomization analysis of >240 POAG and IOP genome-wide association study (GWAS) loci and overlapping expression and splicing quantitative trait loci (e/sQTLs) in 49 GTEx tissues and retina prioritizes causal genes for 60% of loci. These genes are enriched in pathways implicated in extracellular matrix organization, cell adhesion, and vascular development. Analysis of single-nucleus RNA-seq of glaucoma-relevant eye tissues reveals that the POAG and IOP colocalizing genes and genome-wide associations are enriched in specific cell types in the aqueous outflow pathways, retina, optic nerve head, peripapillary sclera, and choroid. This study nominates IOP-dependent and independent regulatory mechanisms, genes, and cell types that may contribute to POAG pathogenesis.

QTL mapping of human retina DNA methylation identifies 87 gene-epigenome interactions in age-related macular degeneration.

DNA methylation (DNAm) provides a crucial epigenetic mark linking genetic variations to environmental influence. We analyzed array-based DNAm profiles of 160 human retinas with co-measured RNA-seq and > 8 million genetic variants, uncovering sites of genetic regulation in cis (37,453 mQTLs and 12,505 eQTLs) and 13,747 eQTMs (DNAm loci affecting gene expression), with over one-third specific to the retina. mQTLs and eQTMs show non-random distribution and enrichment of biological processes related to synapse, mitochondria, and catabolism. Summary data-based Mendelian randomization and colocalization analyses identify 87 target genes where methylation and gene-expression changes likely mediate the genotype effect on age-related macular degeneration (AMD). Integrated pathway analysis reveals epigenetic regulation of immune response and metabolism including the glutathione pathway and glycolysis. Our study thus defines key roles of genetic variations driving methylation changes, prioritizes epigenetic control of gene expression, and suggests frameworks for regulation of AMD pathology by genotype-environment interaction in retina.

Large-scale multitrait genome-wide association analyses identify hundreds of glaucoma risk loci.

Glaucoma, a leading cause of irreversible blindness, is a highly heritable human disease. Previous genome-wide association studies have identified over 100 loci for the most common form, primary open-angle glaucoma. Two key glaucoma-associated traits also show high heritability: intraocular pressure and optic nerve head excavation damage quantified as the vertical cup-to-disc ratio. Here, since much of glaucoma heritability remains unexplained, we conducted a large-scale multitrait genome-wide association study in participants of European ancestry combining primary open-angle glaucoma and its two associated traits (total sample size over 600,000) to substantially improve genetic discovery power (263 loci). We further increased our power by then employing a multiancestry approach, which increased the number of independent risk loci to 312, with the vast majority replicating in a large independent cohort from 23andMe, Inc. (total sample size over 2.8 million; 296 loci replicated at P < 0.05, 240 after Bonferroni correction). Leveraging multiomics datasets, we identified many potential druggable genes, including neuro-protection targets likely to act via the optic nerve, a key advance for glaucoma because all existing drugs only target intraocular pressure. We further used Mendelian randomization and genetic correlation-based approaches to identify novel links to other complex traits, including immune-related diseases such as multiple sclerosis and systemic lupus erythematosus.

Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

Exploiting the GTEx resources to decipher the mechanisms at GWAS loci.

The resources generated by the GTEx consortium offer unprecedented opportunities to advance our understanding of the biology of human diseases. Here, we present an in-depth examination of the phenotypic consequences of transcriptome regulation and a blueprint for the functional interpretation of genome-wide association study-discovered loci. Across a broad set of complex traits and diseases, we demonstrate widespread dose-dependent effects of RNA expression and splicing. We develop a data-driven framework to benchmark methods that prioritize causal genes and find no single approach outperforms the combination of multiple approaches. Using colocalization and association approaches that take into account the observed allelic heterogeneity of gene expression, we propose potential target genes for 47% (2519 out of 5385) of the GWAS loci examined.

Genome-wide meta-analysis identifies 127 open-angle glaucoma loci with consistent effect across ancestries.

Primary open-angle glaucoma (POAG), is a heritable common cause of blindness world-wide. To identify risk loci, we conduct a large multi-ethnic meta-analysis of genome-wide association studies on a total of 34,179 cases and 349,321 controls, identifying 44 previously unreported risk loci and confirming 83 loci that were previously known. The majority of loci have broadly consistent effects across European, Asian and African ancestries. Cross-ancestry data improve fine-mapping of causal variants for several loci. Integration of multiple lines of genetic evidence support the functional relevance of the identified POAG risk loci and highlight potential contributions of several genes to POAG pathogenesis, including SVEP1, RERE, VCAM1, ZNF638, CLIC5, SLC2A12, YAP1, MXRA5, and SMAD6. Several drug compounds targeting POAG risk genes may be potential glaucoma therapeutic candidates.

Cell type-specific genetic regulation of gene expression across human tissues.

The Genotype-Tissue Expression (GTEx) project has identified expression and splicing quantitative trait loci in cis (QTLs) for the majority of genes across a wide range of human tissues. However, the functional characterization of these QTLs has been limited by the heterogeneous cellular composition of GTEx tissue samples. We mapped interactions between computational estimates of cell type abundance and genotype to identify cell type-interaction QTLs for seven cell types and show that cell type-interaction expression QTLs (eQTLs) provide finer resolution to tissue specificity than bulk tissue cis-eQTLs. Analyses of genetic associations with 87 complex traits show a contribution from cell type-interaction QTLs and enables the discovery of hundreds of previously unidentified colocalized loci that are masked in bulk tissue.

The impact of sex on gene expression across human tissues.

Many complex human phenotypes exhibit sex-differentiated characteristics. However, the molecular mechanisms underlying these differences remain largely unknown. We generated a catalog of sex differences in gene expression and in the genetic regulation of gene expression across 44 human tissue sources surveyed by the Genotype-Tissue Expression project (GTEx, v8 release). We demonstrate that sex influences gene expression levels and cellular composition of tissue samples across the human body. A total of 37% of all genes exhibit sex-biased expression in at least one tissue. We identify cis expression quantitative trait loci (eQTLs) with sex-differentiated effects and characterize their cellular origin. By integrating sex-biased eQTLs with genome-wide association study data, we identify 58 gene-trait associations that are driven by genetic regulation of gene expression in a single sex. These findings provide an extensive characterization of sex differences in the human transcriptome and its genetic regulation.

Kinematic behavior of the ankle following malleolar fracture repair in a high-fidelity cadaver model.

BACKGROUND: Previous studies involving axially loaded ankle cadaver specimens undergoing a passive range of motion after fracture have demonstrated rotatory instability patterns consisting of excessive external rotation during plantar flexion. The present study was designed to expand these studies by using a model in which ankle motion is controlled by physiologically accurate motor forces generated through phasic force-couples attached to the muscle-tendon units. METHODS: Eight right unembalmed cadaver feet were tested in a dynamic gait simulator that reproduces the sagittal kinematics of the tibia while applying physiological muscle forces to the tendons of the major extrinsic muscles of the foot. Six-degrees-of-freedom kinematics of the tibia and talus were measured with use of a VICON motion-analysis system. The experimental conditions included all combinations of lateral and medial injury to reproduce the clinical classifications of ankle fracture. Statistical analysis was performed with repeated-measures analyses of variance. RESULTS: The talus of the intact ankles demonstrated coupled external rotation and inversion relative to the tibia as the ankle plantar flexed. Osteotomy of the fibula, simulating a lateral ankle fracture, slightly but significantly increased external rotation and inversion of the talus (p < 0.001), whereas disruption of either the superficial or the deep deltoid ligament increased talar eversion (p < 0.003) and disruption of the deep deltoid ligament increased internal rotation (p < 0.0001). The aberrant motions were corrected by repair of the injured structure. CONCLUSIONS: The predominant coupled rotation of the talus is external rotation associated with plantar flexion. Following progressive ankle destabilization, talar external rotation and inversion increased. CLINICAL RELEVANCE: The clinical decision-making process regarding the treatment of ankle fractures centers on determination of whether the injury is expected to result in abnormal motion, which is thought to predispose to the development of arthritis. The present study demonstrated a remarkable degree of ankle stability during stance phase even when there was severe disruption of medial and lateral structures. This finding suggests that a main determinant of clinical outcome after ankle fracture may be ankle motion during swing phase, when ankle stability is not augmented by the combination of axial loading and active motor control of motion. If swing-phase motion is abnormal, then the ankle may be in a vulnerable position at the point of heel-strike.

Dynamic loading of the plantar aponeurosis in walking.

BACKGROUND: The plantar aponeurosis is known to be a major contributor to arch support, but its role in transferring Achilles tendon loads to the forefoot remains poorly understood. The goal of this study was to increase our understanding of the function of the plantar aponeurosis during gait. We specifically examined the plantar aponeurosis force pattern and its relationship to Achilles tendon forces during simulations of the stance phase of gait in a cadaver model. METHODS: Walking simulations were performed with seven cadaver feet. The movements of the foot and the ground reaction forces during the stance phase were reproduced by prescribing the kinematics of the proximal part of the tibia and applying forces to the tendons of extrinsic foot muscles. A fiberoptic cable was passed through the plantar aponeurosis perpendicular to its loading axis, and raw fiberoptic transducer output, tendon forces applied by the experimental setup, and ground reaction forces were simultaneously recorded during each simulation. A post-experiment calibration related fiberoptic output to plantar aponeurosis force, and linear regression analysis was used to characterize the relationship between Achilles tendon force and plantar aponeurosis tension. RESULTS: Plantar aponeurosis forces gradually increased during stance and peaked in late stance. Maximum tension averaged 96% +/- 36% of body weight. There was a good correlation between plantar aponeurosis tension and Achilles tendon force (r = 0.76). CONCLUSIONS: The plantar aponeurosis transmits large forces between the hindfoot and forefoot during the stance phase of gait. The varying pattern of plantar aponeurosis force and its relationship to Achilles tendon force demonstrates the importance of analyzing the function of the plantar aponeurosis throughout the stance phase of the gait cycle rather than in a static standing position. CLINICAL RELEVANCE: The plantar aponeurosis plays an important role in transmitting Achilles tendon forces to the forefoot in the latter part of the stance phase of walking. Surgical procedures that require the release of this structure may disturb this mechanism and thus compromise efficient propulsion.

Fiberoptic measurement of tendon forces is influenced by skin movement artifact.

Fiberoptic cables have previously been used for tendon force measurements in vivo. To measure forces in the Achilles tendon, a cable is passed mediolaterally through the skin and tendon, transverse to the loading axis. As the tendon is loaded, its fibers compress the cable and modulate the intensity of transmitted light, which can be related to tendon force by an in situ calibration. The relative movement between skin and tendon at the cable entry and exit sites may cause error by bending the cable and thus altering transducer output. Cadaver simulations of walking were conducted to compare fiberoptic measurements of Achilles tendon forces to known loads applied to the tendon by actuators attached in series. Force measurement errors, which were high when the skin was intact (RMS errors 24-81% peak forces), decreased considerably after skin removal (RMS errors 10-33% peak forces). The fiberoptic transducer is a useful tool for measurement of tendon forces in situ under natural loading conditions when skin can be removed, but caution should be exercised during in vivo use of this technique or under circumstances where skin is in contact with the fiberoptic cable at the insertion and exit sites.

Relative motions of the tibia, talus, and calcaneus during the stance phase of gait: a cadaver study.

The motions of the tibia, talus, and calcaneus during walking were analyzed three-dimensionally using a dynamic cadaver model that recreates the stance phase of walking. Rigid marker clusters were attached to each of the three bones, and the rotations of the talus and calcaneus with respect to the tibia and the calcaneus with respect to the talus were analyzed for eight right cadaver feet. The talus rotated primarily in plantarflexion/dorsiflexion about the talocrural joint, with an average range of 18 degrees +/- 4.7 degrees. The calcaneus began in inversion and internal rotation with respect to the tibia, moved into the neutral position at 28% of the stance phase and rotated primarily in plantarflexion from that point onward. Rotation of the calcaneus with respect to the talus at the subtalar joint occurred about all three axes, with approximately 5 degrees of relative dorsiflexion and 7 degrees of relative internal rotation. After 25% of stance, the talus and calcaneus moved together as one body into plantarflexion, providing a rigid lever as toe-off was approached.

The effect of ankle injury on subtalar motion.

BACKGROUND: Injuries to the medial and lateral ankle ligaments have been implicated in subtalar joint instability. Lateral injury increased subtalar joint varus and anterior translation, while deltoid injury increased external rotation and valgus in studies using static, non-physiologic testing. METHODS: The current study employed a physiologically accurate ankle model using phasic force-couples attached to the muscle-tendon units to reproduce ankle motion. Six-degree-of-freedom kinematics of the tibia, talus, and calcaneus were measured using a VICON motion analysis system under the following experimental conditions: 1) intact ligaments 2) complete lateral ligament injury with subsequent repair, 3) superficial deltoid injury with subsequent repair, and 4) deep deltoid injury without repair in eight harvested lower extremities. Statistical analysis was by repeated measures analyses of variance. RESULTS: At heel-strike, the subtalar joint is in internal rotation, dorsiflexion, and varus. As the leg progresses to foot-flat, there is external rotation, plantarflexion, and valgus rotation. From foot-flat to heel-rise, there is little subtalar joint motion, while at toe-off, there is slight internal rotation, dorsiflexion, and varus rotation. The total rotations amounted to 9.0 degrees (SD 5.0 degrees) external rotation, 6.1 degrees (SD 2.5 degrees) plantarflexion, and 7.8 degrees (SD 5.5 degrees) valgus. Disruption of the superficial deltoid increased plantarflexion (p < .001) and valgus (p < .05). The additional lateral injury increased both external rotation (p < .001) and valgus (p < .02). Lateral injury alone had no significant effect on subtalar joint motion. CONCLUSION: Unlike most previous reports, this study showed no significant influence of isolated lateral ankle injury on subtalar joint motion, probably because the current study examined subtalar joint motion under physiologic loading and motion rather than by static stress testing. This calls into question the relevance of static stress testing to the in situ function of the subtalar joint. The increased external rotation and valgus seen with deltoid injury in the current study is consistent with previous reports.

A dynamic cadaver model of the stance phase of gait: performance characteristics and kinetic validation.

OBJECTIVE: This study was undertaken to evaluate the performance of a new dynamic laboratory model of the stance phase of gait. DESIGN: Five cadaver feet were repetitively tested in the apparatus. BACKGROUND: Typical biomechanical investigations of cadaver feet simply place a static load on the tibia. The present system was designed to better simulate the changing in-vivo loading environment of the foot and ankle during gait. METHODS: The device mimics the behavior of the tibia, foot, and ankle from heel-strike to toe-off by reproducing the physiologic actions of five extrinsic foot muscles and physiologic motion at the proximal tibia. To verify its utility, cadaver gait simulations were conducted while measuring applied muscle forces, ground reaction forces, and plantar pressures. RESULTS: Dynamic muscle forces were consistently delivered to within 10% of pre-programmed values. Dynamic measurements of ground reaction forces and plantar pressure were similar to those measured in healthy human subjects. Peak vertical (y), foreaft (x) and medio-lateral (z) forces were 110, 18, and 4% of body weight respectively. Compressive force in the tibial shaft reached 410% of body weight. RELEVANCE: Cadaver studies have greatly enhanced our understanding of normal and pathologic foot function, but are often limited by over-simplified loading conditions. The apparatus presented here accurately reproduces the in-vivo loading environment and provides a powerful investigational tool for the study of foot and ankle function. With this device, musculoskeletal structures can be examined in detail under biomechanical conditions similar to those they experience in life.

Development of an animal model of acetabular fractures.

A closed intraarticular fracture is a complex injury that consists of a physical disruption of the subchondral bone and articular surface, and an impaction injury to the articular surface that occurs at the time of the fracture. Few experimental models have been able to incorporate the elements of displaced articular fractures and blunt impaction injury to the articular surface. This work details the initial stages of an attempt to develop such model. Using a model of a dorsal wall fracture of the acetabulum in a goat, we have developed a bench-top method for assessing articular contact stress. Additionally, preliminary in vivo survival data are presented.