Embryos assist morphogenesis of others through calcium and ATP signaling mechanisms in collective teratogen resistance.

Information for organismal patterning can come from a variety of sources. We investigate the possibility that instructive influences for normal embryonic development are provided not only at the level of cells within the embryo, but also via interactions between embryos. To explore this, we challenge groups of embryos with disruptors of normal development while varying group size. Here, we show that Xenopus laevis embryos are much more sensitive to a diverse set of chemical and molecular-biological perturbations when allowed to develop alone or in small groups, than in large groups. Keeping per-embryo exposure constant, we find that increasing the number of exposed embryos in a cohort increases the rate of survival while incidence of defects decreases. This inter-embryo assistance effect is mediated by short-range diffusible signals and involves the P2 ATP receptor. Our data and computational model emphasize that morphogenesis is a collective phenomenon not only at the level of cells, but also of whole bodies, and that cohort size is a crucial variable in studies of ecotoxicology, teratogenesis, and developmental plasticity.

Target-agnostic drug prediction integrated with medical record analysis uncovers differential associations of statins with increased survival in COVID-19 patients.

Drug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. A Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. Simvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

Enhancers of Host Immune Tolerance to Bacterial Infection Discovered Using Linked Computational and Experimental Approaches.

Current therapeutic strategies against bacterial infections focus on reduction of pathogen load using antibiotics; however, stimulation of host tolerance to infection in the presence of pathogens might offer an alternative approach. Computational transcriptomics and Xenopus laevis embryos are used to discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus exhibits natural tolerance to Acinetobacter baumanii, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae bacteria, whereas Aeromonas hydrophila and Pseudomonas aeruginosa produce lethal infections. Transcriptional profiling leads to definition of a 20-gene signature that discriminates between tolerant and susceptible states, as well as identification of a more active tolerance response to gram negative compared to gram positive bacteria. Gene pathways associated with active tolerance in Xenopus, including some involved in metal ion binding and hypoxia, are found to be conserved across species, including mammals, and administration of a metal chelator (deferoxamine) or a HIF-1α agonist (1,4-DPCA) in embryos infected with lethal A. hydrophila increased survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with computational multiomics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infections.

Studying Protista WBR and Repair Using Physarum polycephalum.

Physarum polycephalum is a protist slime mould that exhibits a high degree of responsiveness to its environment through a complex network of tubes and cytoskeletal components that coordinate behavior across its unicellular, multinucleated body. Physarum has been used to study decision making, problem solving, and mechanosensation in aneural biological systems. The robust generative and repair capacities of Physarum also enable the study of whole-body regeneration within a relatively simple model system. Here we describe methods for growing, imaging, quantifying, and sampling Physarum that are adapted for investigating regeneration and repair.

Target-agnostic drug prediction integrated with medical record analysis uncovers differential associations of statins with increased survival in COVID-19 patients.

Importance: Drug repurposing requires distinguishing established drug class targets from novel molecule-specific mechanisms and rapidly derisking their therapeutic potential in a time-critical manner, particularly in a pandemic scenario. In response to the challenge to rapidly identify treatment options for COVID-19, several studies reported that statins, as a drug class, reduce mortality in these patients. However, it is unknown if different statins exhibit consistent function or may have varying therapeutic benefit. Objectives: To test if different statins differ in their ability to exert protective effects based on molecular computational predictions and electronic medical record analysis. Main Outcomes and Measures: A Bayesian network tool was used to predict drugs that shift the host transcriptomic response to SARS-CoV-2 infection towards a healthy state. Drugs were predicted using 14 RNA-sequencing datasets from 72 autopsy tissues and 465 COVID-19 patient samples or from cultured human cells and organoids infected with SARS-CoV-2, with a total of 2,436 drugs investigated. Top drug predictions included statins, which were then assessed using electronic medical records containing over 4,000 COVID-19 patients on statins to determine mortality risk in patients prescribed specific statins versus untreated matched controls. The same drugs were tested in Vero E6 cells infected with SARS-CoV-2 and human endothelial cells infected with a related OC43 coronavirus. Results: Simvastatin was among the most highly predicted compounds (14/14 datasets) and five other statins, including atorvastatin, were predicted to be active in > 50% of analyses. Analysis of the clinical database revealed that reduced mortality risk was only observed in COVID-19 patients prescribed a subset of statins, including simvastatin and atorvastatin. In vitro testing of SARS-CoV-2 infected cells revealed simvastatin to be a potent direct inhibitor whereas most other statins were less effective. Simvastatin also inhibited OC43 infection and reduced cytokine production in endothelial cells. Conclusions and Relevance: Different statins may differ in their ability to sustain the lives of COVID-19 patients despite having a shared drug target and lipid-modifying mechanism of action. These findings highlight the value of target-agnostic drug prediction coupled with patient databases to identify and clinically evaluate non-obvious mechanisms and derisk and accelerate drug repurposing opportunities.

Acute multidrug delivery via a wearable bioreactor facilitates long-term limb regeneration and functional recovery in adult Xenopus laevis.

Limb regeneration is a frontier in biomedical science. Identifying triggers of innate morphogenetic responses in vivo to induce the growth of healthy patterned tissue would address the needs of millions of patients, from diabetics to victims of trauma. Organisms such as Xenopus laevis-whose limited regenerative capacities in adulthood mirror those of humans-are important models with which to test interventions that can restore form and function. Here, we demonstrate long-term (18 months) regrowth, marked tissue repatterning, and functional restoration of an amputated X. laevis hindlimb following a 24-hour exposure to a multidrug, pro-regenerative treatment delivered by a wearable bioreactor. Regenerated tissues composed of skin, bone, vasculature, and nerves significantly exceeded the complexity and sensorimotor capacities of untreated and control animals' hypomorphic spikes. RNA sequencing of early tissue buds revealed activation of developmental pathways such as Wnt/ß-catenin, TGF-ß, hedgehog, and Notch. These data demonstrate the successful "kickstarting" of endogenous regenerative pathways in a vertebrate model.

Painful temporomandibular joint overloading induces structural remodeling in the pericellular matrix of that joint's chondrocytes.

Mechanical stress to the temporomandibular joint (TMJ) is an important factor in cartilage degeneration, with both clinical and preclinical studies suggesting that repeated TMJ overloading could contribute to pain, inflammation, and/or structural damage in the joint. However, the relationship between pain severity and early signs of cartilage matrix microstructural dysregulation is not understood, limiting the advancement of diagnoses and treatments for temporomandibular joint-osteoarthritis (TMJ-OA). Changes in the pericellular matrix (PCM) surrounding chondrocytes may be early indicators of OA. A rat model of TMJ pain induced by repeated jaw loading (1 h/day for 7 days) was used to compare the extent of PCM modulation for different loading magnitudes with distinct pain profiles (3.5N-persistent pain, 2N-resolving pain, or unloaded controls-no pain) and macrostructural changes previously indicated by Mankin scoring. Expression of PCM structural molecules, collagen VI and aggrecan NITEGE neo-epitope, were evaluated at Day 15 by immunohistochemistry within TMJ fibrocartilage and compared between pain conditions. Pericellular collagen VI levels increased at Day 15 in both the 2N (p = 0.003) and 3.5N (p = 0.042) conditions compared to unloaded controls. PCM width expanded to a similar extent for both loading conditions at Day 15 (2N, p < 0.001; 3.5N, p = 0.002). Neo-epitope expression increased in the 3.5N group over levels in the 2N group (p = 0.041), indicating pericellular changes that were not identified in the same groups by Mankin scoring of the pericellular region. Although remodeling occurs in both pain conditions, the presence of pericellular catabolic neo-epitopes may be involved in the macrostructural changes and behavioral sensitivity observed in persistent TMJ pain.

Increased substance P and synaptic remodeling occur in the trigeminal sensory system with sustained osteoarthritic temporomandibular joint sensitivity.

INTRODUCTION: Temporomandibular joint (TMJ) pain is among the most prevalent musculoskeletal conditions and can result from atypical joint loading. Although TMJ pain is typically self-resolving, 15% of patients develop chronic TMJ pain that is recalcitrant to therapy and may be attributed to changes in pain processing centers. Although TMJ overloading induces pain and osteoarthritis, whether neuronal modifications in the trigeminal sensory system contribute to persistent TMJ pain is unknown. OBJECTIVE: This study investigates changes in excitatory neuropeptides and synaptic transmission proteins in cases of transient and persistent TMJ sensitivity in a rat model. METHODS: Rats underwent repeated jaw loading that produces transient (2N-load) or persistent (3.5N-load) sensitivity. In both groups, immunolabeling was used to assess substance P in the spinal trigeminal nucleus caudalis (Sp5C) and glutamate transporter 1 in the ventroposteriomedial thalamus early after loading. Synaptosomal Western blots were used to measure synaptic proteins in the caudal medulla and thalamus at a later time after loading. RESULTS: Substance P increases transiently in the Sp5C early after loading that induces persistent sensitivity. However, glutamate transporter 1 is unchanged in the ventroposteriomedial thalamus. At a later time, synaptosomal Western blots show loss of the presynaptic tethering protein, synapsin, and the inhibitory scaffolding protein, gephyrin, in the thalamus with persistent, but not transient, sensitivity. No changes are identified in synapsin, phosphorylated synapsin, homer, or gephyrin in the caudal medulla. CONCLUSIONS: Substance P in the Sp5C and later loss of inhibitory synapses in the thalamus likely contribute to, or indicate, persistent TMJ pain.

Early changes in brain network topology and activation of affective pathways predict persistent pain in the rat.

Adaptations in brain communication are associated with multiple pain disorders and are hypothesized to promote the transition from acute to chronic pain. Despite known increases in brain synaptic activity, it is unknown if and how changes in pathways and networks contribute to persistent pain. A tunable rat model that induces transient or persistent temporomandibular joint pain was used to characterize brain network and subcircuit changes when sensitivity is detected in both transient and persistent pain groups and later when sensitivity is present only for the persistent pain group. Brain activity was measured by F-FDG positron emission tomography imaging and used to construct intersubject correlation networks; network connectivity distributions, diagnostics, and community structure were assessed. Activation of subcircuits was tested by structural equation modeling. Findings reveal differences in the brain networks at day 7 between the persistent and transient pain groups, a time when peripheral sensitivity is detected in both groups, but spontaneous pain occurs only in the persistent pain group. At day 7, increased (P ≤ 0.01) clustering, node strength, network segregation, and activation of prefrontal-limbic pathways are observed only in the group that develops persistent pain. Later, increased clustering and node strength are more pronounced with persistent pain, particularly within the limbic system, and decrease when pain resolves. Pretreatment with intra-articular etanercept to attenuate pain confirms that these adaptations are associated with pain onset. Results suggest that early and sustained brain changes can differentiate persistent and transient pain, implying they could be useful as prognostic biomarkers for persistent pain and in identifying therapeutic targets.

Intra-articular etanercept attenuates pain and hypoxia from TMJ loading in the rat.

Mechanical overloading of the temporomandibular joint (TMJ) and biochemical changes, like inflammation and hypoxia, contribute to cartilage degeneration and pain associated with osteoarthritis (OA). Yet, how overloading contributes to early dysregulation of chondrocytes is not understood, limiting the development of diagnostics and treatments for TMJ OA. Hypoxia-inducible factors (HIF)-1α/2α in chondrocytes were evaluated at Days 8 and 15 in a rat TMJ pain model induced by jaw loading (1 h/day for 7 days) using immunohistochemistry and compared between cases that induce persistent (3.5 N), acute (2 N), or no (0 N) sensitivity. Hypoxia was measured on Day 8 by immunolabeling of the tracer EF5 and 18 F-EF5 PET imaging. To assess the role of tumor necrosis factor (TNF) in painful TMJ loading, intra-articular etanercept was given before loading. Orofacial sensitivity was evaluated during and after loading. Facial grimace, TNF-α, HIF-2α, and hypoxia levels in the TMJ were measured after loading. HIF-2α was elevated (P = .03) after 3.5 N loading at Day 8, but HIF-1α was unchanged. EF5 uptake increased on Day 8 in the 3.5 N group (P < .048) by tissue assay and 18 F-EF5 PET. At Day 8, both HIF-2α (P = .01) and EF5 uptake (P = .005) were correlated with loading magnitude. Etanercept attenuated sensitivity (P < .01) and the facial grimace on Day 7 (P = .01). It also reduced (P < .01) HIF-2α and EF5 uptake on Day 8; but TNF-α levels were not different from controls at that time. Findings suggest that TMJ loading that induces persistent sensitivity upregulates the catabolic factor HIF-2α and reduces oxygen levels in the cartilage, which may be TNF-driven.

Lower back pain and healthy subjects exhibit distinct lower limb perturbation response strategies: A preliminary study.

BACKGROUND: It is hypothesized that inherent differences in movement strategies exist between control subjects and those with a history of lower back pain (LBP). Previous motion analysis studies focus primarily on tracking spinal movements, neglecting the connection between the lower limbs and spinal function. Lack of knowledge surrounding the functional implications of LBP may explain the diversity in success from general treatments currently offered to LBP patients. OBJECTIVE: This pilot study evaluated the response of healthy controls and individuals with a history of LBP (hLBP) to a postural disturbance. METHODS: Volunteers (n= 26) were asked to maintain standing balance in response to repeated balance disturbances delivered via a perturbation platform while both kinematic and electromyographic data were recorded from the trunk, pelvis, and lower limb. RESULTS: The healthy cohort utilized an upper body-focused strategy for balance control, with substantial activation of the external oblique muscles. The hLBP cohort implemented a lower limb-focused strategy, relying on activation of the semitendinosus and soleus muscles. No significant differences in joint range of motion were identified. CONCLUSIONS: These findings suggest that particular reactive movement patterns may indicate muscular deficits in subjects with hLBP. Identification of these deficits may aid in developing specific rehabilitation programs to prevent future LBP recurrence.

Grading facial expression is a sensitive means to detect grimace differences in orofacial pain in a rat model.

Although pre-clinical models of pain are useful for defining relationships between biological mechanisms and pain, common methods testing peripheral sensitivity do not translate to the human pain experience. Facial grimace scales evaluate affective pain levels in rodent models by capturing and scoring spontaneous facial expression. But, the Rat Grimace Scale (RGS) has not assessed the common disorder of temporomandibular joint (TMJ) pain. A rat model of TMJ pain induced by jaw loading (1 hr/day for 7 days) was used to investigate the time course of RGS scores and compare them between different loading magnitudes with distinct peripheral sensitivity profiles (0N-no sensitivity, 2N-acute sensitivity, 3.5N-persistent sensitivity). In the 3.5N group, RGS is elevated over baseline during the loading period and one day after loading and is correlated with peripheral sensitivity (ρ = -0.48, p = 0.002). However, RGS is not elevated later when that group exhibits peripheral sensitivity and moderate TMJ condylar cartilage degeneration. Acutely, RGS is elevated in the 3.5N loading group over the other loading groups (p < 0.001). These findings suggest that RGS is an effective tool for detecting spontaneous TMJ pain and that spontaneous pain is detectable in rats that develop persistent TMJ sensitivity, but not in rats with acute resolving sensitivity.

Inter-subject FDG PET Brain Networks Exhibit Multi-scale Community Structure with Different Normalization Techniques.

Inter-subject networks are used to model correlations between brain regions and are particularly useful for metabolic imaging techniques, like 18F-2-deoxy-2-(18F)fluoro-D-glucose (FDG) positron emission tomography (PET). Since FDG PET typically produces a single image, correlations cannot be calculated over time. Little focus has been placed on the basic properties of inter-subject networks and if they are affected by group size and image normalization. FDG PET images were acquired from rats (n = 18), normalized by whole brain, visual cortex, or cerebellar FDG uptake, and used to construct correlation matrices. Group size effects on network stability were investigated by systematically adding rats and evaluating local network connectivity (node strength and clustering coefficient). Modularity and community structure were also evaluated in the differently normalized networks to assess meso-scale network relationships. Local network properties are stable regardless of normalization region for groups of at least 10. Whole brain-normalized networks are more modular than visual cortex- or cerebellum-normalized network (p < 0.00001); however, community structure is similar at network resolutions where modularity differs most between brain and randomized networks. Hierarchical analysis reveals consistent modules at different scales and clustering of spatially-proximate brain regions. Findings suggest inter-subject FDG PET networks are stable for reasonable group sizes and exhibit multi-scale modularity.

The Interface of Mechanics and Nociception in Joint Pathophysiology: Insights From the Facet and Temporomandibular Joints.

Chronic joint pain is a widespread problem that frequently occurs with aging and trauma. Pain occurs most often in synovial joints, the body's load bearing joints. The mechanical and molecular mechanisms contributing to synovial joint pain are reviewed using two examples, the cervical spinal facet joints and the temporomandibular joint (TMJ). Although much work has focused on the macroscale mechanics of joints in health and disease, the combined influence of tissue mechanics, molecular processes, and nociception in joint pain has only recently become a focus. Trauma and repeated loading can induce structural and biochemical changes in joints, altering their microenvironment and modifying the biomechanics of their constitutive tissues, which themselves are innervated. Peripheral pain sensors can become activated in response to changes in the joint microenvironment and relay pain signals to the spinal cord and brain where pain is processed and perceived. In some cases, pain circuitry is permanently changed, which may be a potential mechanism for sustained joint pain. However, it is most likely that alterations in both the joint microenvironment and the central nervous system (CNS) contribute to chronic pain. As such, the challenge of treating joint pain and degeneration is temporally and spatially complicated. This review summarizes anatomy, physiology, and pathophysiology of these joints and the sensory pain relays. Pain pathways are postulated to be sensitized by many factors, including degeneration and biochemical priming, with effects on thresholds for mechanical injury and/or dysfunction. Initiators of joint pain are discussed in the context of clinical challenges including the diagnosis and treatment of pain.

Rentian scaling for the measurement of optimal embedding of complex networks into physical space.

The London Underground is one of the largest, oldest and most widely used systems of public transit in the world. Transportation in London is constantly challenged to expand and adapt its system to meet the changing requirements of London's populace while maintaining a cost-effective and efficient network. Previous studies have described this system using concepts from graph theory, reporting network diagnostics and core-periphery architecture. These studies provide information about the basic structure and efficiency of this network; however, the question of system optimization in the context of evolving demands is seldom investigated. In this paper we examined the cost effectiveness of the topological-physical embedding of the Tube using estimations of the topological dimension, wiring length and Rentian scaling, an isometric scaling relationship between the number of elements and connections in a system. We measured these properties in both two- and three-dimensional embeddings of the networks into Euclidean space, as well as between two time points, and across the densely interconnected core and sparsely interconnected periphery. While the two- and three-dimensional representations of the present-day Tube exhibit Rentian scaling relationships between nodes and edges of the system, the overall network is approximately cost-efficiently embedded into its physical environment in two dimensions, but not in three. We further investigated a notable disparity in the topology of the network's local core versus its more extended periphery, suggesting an underlying relationship between meso-scale structure and physical embedding. The collective findings from this study, including changes in Rentian scaling over time, provide evidence for differential embedding efficiency in planned versus self-organized networks. These findings suggest that concepts of optimal physical embedding can be applied more broadly to other physical systems whose links are embedded in a well-defined space, and whose topology is constrained by a cost function that minimizes link lengths within that space.