Search

Extracellular matrix (ECM) interactions regulate both the cell transcriptome and proteome, thereby determining cell fate. Traumatic heterotopic ossification (HO) is a disorder characterized by aberrant mesenchymal lineage (MLin) cell differentiation, forming bone within soft tissues of the musculoskeletal system following traumatic injury. Recent work has shown that HO is influenced by ECM-MLin cell receptor signaling, but how ECM binding affects cellular outcomes remains unclear. Using time course transcriptomic and proteomic analyses, we identified discoidin domain receptor 2 (DDR2), a cell surface receptor for fibrillar collagen, as a key MLin cell regulator in HO formation. Inhibition of DDR2 signaling, through either constitutive or conditional Ddr2 deletion or pharmaceutical inhibition, reduced HO formation in mice. Mechanistically, DDR2 perturbation alters focal adhesion orientation and subsequent matrix organization, modulating Focal Adhesion Kinase (FAK) and Yes1 Associated Transcriptional Regulator and WW Domain Containing Transcription Regulator 1 (YAP/TAZ)–mediated MLin cell signaling. Hence, ECM-DDR2 interactions are critical in driving HO and could serve as a previously unknown therapeutic target for treating this disease process.

Purpose: Cells recognize mechanical cues from the extracellular environment through interactions with matrix proteins, such as collagen. Following injury, fibrotic deposition of collagen can lead to aberrant cell differentiation and failed healing. Aligned collagen matrices can drive PDGFRα+ mesenchymal progenitor cells (MPCs) towards osteochondral lineages, leading to heterotopic ossification (HO). Previous work has shown that reducing mechanical strain by immobilizing a limb prevents the formation of an aligned extracellular matrix (ECM), leading to altered MPC differentiation, however, the cellular mechanisms of matrix reorganization following injury remain unclear. Using novel bioinformatics approach, we identified discoidin domain receptor 2 (DDR2) as a key MPC specific tyrosine kinase receptor that interacts with the fibrillar collagen matrix. We hypothesized that DDR2 activation leads to regulation of ECM alignment and therefore serves as a novel upstream regulator of mechanotransductive signaling following musculoskeletal repair. Methods: Heterotopic ossification was induced using a proven mouse model of 30% total body surface area burn with concurrent Achilles’ transection in Ddr2 deletion (Ddr2slie/slie) and littermate control mice on the C57/BL6J background. In separate experiments, ankle joint immobilizers were placed on injured mice for 1, 2 or 3 weeks after burn/tenotomy. Tissue from Ddr2 deleted, immobilized and mobile control mice were harvested for immunofluorescence histology, second harmonic generation of collagen fiber histology at 1, 3 and 9 weeks after injury (SHG) (n=4/group). HO site tissue was also harvested prior to injury and at 1 and 6 weeks after burn/tenotomy and processed for Single-Cell RNA sequencing (scRNA seq) and single nucleus assay for transposase-accessible chromatin (snATAC) using 10X Sequencing and downstream analysis using Seurat 4.0.1 and Signac 3.1.5. Results: Single-cell RNA sequencing showed that genes for collagen type 1 and 3 and Ddr2 are highly upregulated in MPCs following injury. SnATAC showed increased open chromatin reads in the promoter of MPCs following injury (not shown). Utilizing SHG, we found that the collagen matrix is aligned at three-weeks following injury, but one week of limb immobilization was enough to prevent aligned collagen. The active phosphorylated form of DDR2 (pDDR2) was decreased in PDGFRα+ MPCs within immobilized samples, suggesting that DDR2 may affect matrix alignment. Ddr2slie/slie had more disorganized collagen matrix nine weeks following injury and significantly reduced HO. Conclusions: This is the first work to alter collagen matrix organization by genetic modification and mobilization protocols and identifies DDR2 as a critical upstream receptor in matrix organization and aberrant progenitor differentiation following injury. DDR2 antagonism could have therapeutic potential in preventing heterotopic ossification following traumatic injury.

Approximately 40 million adults use marijuana annually, making marijuana one of the most commonly used substances in the United States (SAMHSA, 2019). Men and emerging adults (ages 18-25) report higher prevalence of cannabis use disorders (CUDs) relative to women and older adults (CBHSQ, 2015; Khan et al., 2013). More frequent marijuana use is associated with greater likelihood of suicidal ideation (Ilgen et al., 2009), and past year use in emerging adults is associated with future suicide attempts (Pedersen, 2008). Similar to correlates of marijuana use, emerging adults and men have higher rates of suicidality (SAMHSA, 2019; Krug et al., 2002). Limited research has tested gender and age differences in comorbid CUDs and suicidality. The current study evaluated gender and age differences in CUDs only, suicidality only, or comorbid CUDs and suicidality in a national sample of adults. We hypothesized that men and emerging adults would be over-represented in comorbid CUDs and suicidality and CUDs only groups. Data were from four consecutive years (2015-2018) of the National Survey of Drug Use and Heath. Multinomial logistic regressions tested gender and age differences in adults with DSM-IV cannabis abuse or dependence (CUDs) only, suicidality only, and comorbid CUDs and suicidality, all compared to adults with neither CUDs or suicidality. Four separate regressions were conducted for passive suicidal ideation, active suicidal ideation, suicide planning, and suicide attempts. Gender was coded as male or female. Age groups were 18–25, 26–34, 35–49, and 50 years or older. Analyses controlled for survey year, race/ethnicity, sexual orientation, education, household income, past year major depressive episode, past year DSM-IV alcohol abuse or dependence, and past year illicit drug abuse or dependence other than CUDs. Men disproportionately reported CUDs only (ORs=1.73-2.19, p

Summary Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreERT2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion., Graphical Abstract, Highlights • Lineage tracing, single-cell RNA-seq and single cell ATAC enable cell specific analysis of in vivo cell fate • Hoxa11 lineage marks distinct mesenchymal precursors in the zeugopod • Hoxa11 lineage mesenchymal precursors undergo an aberrant cell fate change towards ectopic bone and cartilage, Here, Pagani et al. use the Hoxa11 patterning gene, expressed exclusively in the limb zeugopod, to demonstrate by bioinformatic single cell transcriptomic, epigenomic analyses, and ex vivo confocal microscopy that extra-skeletal cells originating from Hoxa11 lineage cells are a population of mesenchymal cells with the ability to undergo aberrant differentiation towards chondrocytes, osteoblasts or adipocytes after injury

Purpose: Repair of distal phalanx amputation is widely studied and thought to occur through a regenerative process. Single-cell RNA sequencing (scRNA) has greatly improved the ability to characterize the cells that are involved in regeneration and abnormal healing of bone and soft tissue. Given the disorganized architecture of digit tip “regenerated” bone and its common progenitor cell to the pathologic process of heterotopic ossification, we hypothesized that digit tip repair after injury follows a program of aberrant wound repair rather than true regeneration of normal bone tissue. Methods: ScRNA data of mouse digit tip amputation (DTA) and embryonic limb development (EMB) was downloaded from NBCI GEO data base ascension number GSE135985. Additionally, scRNA data from heterotopic ossification (HO) was downloaded from GSE126060. HO, DTA, and EMB datasets were clustered individually. HO was merged and aligned with EMB and DT in separate analyses. Cells expressing the mesenchymal progenitor cell (MPC) marker Pdgfra, which we have shown to differentiate into HO and others have shown to differentiate into bone after DTA, were identified and analyzed in the HO-DTA and HO-EMB datasets. Monocle 2, Seurat 3.1.1, and Pearson correlation analysis were used for analysis and visualization. Immunostaining for markers present in DTA (ACAN, FBN-2, LTBP2) was performed on sections collected from a murine model of traumatic HO in Pdgfra-CreER;TdTomato mice induced with tamoxifen one-week prior to HO-forming injury. Results: MPC clusters from DTA and HO and from EMB and HO were aligned and projected using UMAP to compare transcription profiles. Across the timepoints, MPCs in the DTA and HO occupy similar regions of the UMAP plot, while MPCs in EMB and HO occupy largely independent regions, with only overlap at the latest time points. Using Monocle 2, cells were placed on a virtual timeline based on similarities in transcription. MPCs from DTA and HO meet at the same location on the trajectory following injury. In the EMB to HO comparison, cells only occupy similar regions of the trajectory at post-natal day 3 suggesting that bone is formed by different pathways. To understand correlations between time points, transcriptomes were also compared by Pearson analysis. MPCs following HO and DTA injuries occupy similar regions of the plot while MPCs in EMB and HO only occupy similar regions of the correlation plot at P3. Markers previously identified to be upregulated in MPCs in DTA were also upregulated in MPCs in HO by both scRNA and immunofluorescent histology. Conclusions: Cells responsible for bone repair following DTA show similar transcriptional profiles as MPCs in traumatic HO and dissimilar to MPCs in EMB. Furthermore, MPCs in both digit tip repair and HO follow trajectories that do not overlap with embryonic limb development. Thus, we demonstrate for the first time, that the process following DTA is not truly regeneration and follows programs of aberrant repair as seen in HO.

While modularity is thought to be central for the evolution of complexity and evolvability, it remains unclear how systems boot-strap themselves into modularity from random or fully integrated starting conditions. Clune et al. (2013) suggested that a positive correlation between sparsity and modularity is the prime cause of this transition. We sought to test the generality of this modularity-sparsity hypothesis by testing it for the first time in physically embodied robots. A population of ten Tadros — autonomous, surface-swimming robots propelled by a flapping tail — was used. Individuals varied only in the structure of their neural net control, a 2 x 6 x 2 network with recurrence in the hidden layer. Each of the 60 possible connections was coded in the genome, and could achieve one of three states: -1, 0, 1. Inputs were two light-dependent resistors and outputs were two motor control variables to the flapping tail, one for the frequency of the flapping and the other for the turning offset. Each Tadro was tested separately in a circular tank lit by a single overhead light source. Fitness was the amount of light gathered by a vertically oriented sensor that was disconnected from the controller net. Reproduction was asexual, with the top performer cloned and then all individuals entered into a roulette wheel selection process, with genomes mutated to create the offspring. The starting population of networks was randomly generated. Over ten generations, the population’s mean fitness increased two-fold. This evolution occurred in spite of an unintentional integer overflow problem in recurrent nodes in the hidden layer that caused outputs to oscillate. Our investigation of the oscillatory behavior showed that the mutual information of inputs and outputs was sufficient for the reactive behaviors observed. While we had predicted that both modularity and sparsity would follow the same trend as fitness, neither did so. Instead, selection gradients within each generation showed that selection directly targeted sparsity of the connections to the motor outputs. Modularity, while not directly targeted, was correlated with sparsity, and hence was an indirect target of selection, its evolution a by-product of its correlation with sparsity.

Random recombination in evolutionary algorithms can be counterproductive in systems that evolve increasing modularity, because such operators do not preserve community structures during their development. Partly because of this, methods have been proposed that derandomize recombination by placing potential crossover locations under evolutionary control. Since crossover is likely to be particularly useful when genetic material that generates incipient phenotype modules is recombined, there may be an advantage to seeking such modularity directly in the phenotype and probabilistically focusing recombination at such "hotspot" locations. Here we show that such phenotypically-aware crossover operators can outcompete random or evolved crossover points as the size of the system being evolved grows. As this crossover operator can be viewed as epigenetic, and as epigenetic processes seem to be common in biological systems, other such epigenetic mechanisms may further improve future evolutionary algorithms.

To understand how to modulate the behavior of underwater swimmers propelled by multiple appendages, we conducted surge maneuver experiments on our biologically-inspired robot, Madeleine. Robot Madeleine is a self-contained, self-propelled underwater vehicle with onboard processor, sensors and power supply. Madeleine's four flippers, oscillating in pitch, can be independently controlled, allowing us to test the impact of flipper phase on performance. We tested eight gaits, four four-flippered and four two-flippered. Gaits were selected to vary the phase, at either 0 or pi rad, between flippers on one side, producing a fore-aft interaction, or flippers on opposite sides, producing a port-starboard interaction. During rapid starting, top-speed cruising, and powered stopping, the power draw, linear acceleration and position of Madeleine were measured. Four-flippered gaits produced higher peak start accelerations than two, but did so with added power draw. During cruising, peak speeds did not vary by flipper number, but power consumption was double in four flippers compared to that of two flippers. Cost of transport (J N(-1) m(-1)) was lower for two-flippered gaits and compares favorably with that of aquatic tetrapods. Two four-flippered gaits produce the highest surge scope, a measure of the difference in peak forward and reverse acceleration. Thus four flippers produce superior surge behavior but do so at high cost; two flippers serve well for lost-cost cruising.

An algorithm was developed to denoise speech presented in matching speech‐spectrum noise by estimating the ideal binary mask (IBM) that retains the time‐frequency (T‐F) regions with favorable local signal‐to‐noise ratio (SNR) and removes the remaining regions [Hu et al., in Proceedings of the 4th SAPA Workshop, Interspeech 2008]. With this method sentence intelligibility was tested in 20 elderly and 20 normal‐hearing young listeners. The elderly group had, at worst, moderate SNHL between 0.5 and 4 kHz. Speech material consisted of IEEE sentences spoken by a single female talker. Intelligibility was tested at two SNRs in the elderly (−2 and −4 dB) and three SNRs in the young group (−4, −6, and −8 dB). Subjects listened to 50 sentences at each SNR in three conditions: denoising by the estimated IBM by the algorithm, denoising by the IBM calculated from premixed signals, and the sentences presented in the original noise without processing. With this last condition taken as the baseline, both keyword‐ and phonemic intelligibility of the sentences processed with the algorithm showed a slight improvement for the young but no improvement for the elderly listeners, while both groups performed substantially better than the baseline in the IBM condition. [Work supported by the VA Medical Research.]