CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

Osmotic Pressure as Driving Force for Reducing the Size of Nanoparticles in Emulsions.

We describe here a method to decrease particle size of nanoparticles synthesized by miniemulsion polymerization. Small nanoparticles or nanocapsules were obtained by generating an osmotic pressure to induce the diffusion of monomer molecules from the dispersed phase of a miniemulsion before polymerization to an upper oil layer. The size reduction is dependent on the difference in concentration of monomer in the dispersed phase and in the upper oil layer and on the solubility of the monomer in water. By labeling the emulsion droplets with a copolymer of stearyl methacrylate and a polymerizable dye, we demonstrated that the migration of the monomer to the upper hexadecane layer relied on molecular diffusion rather than diffusion of monomer droplets to the oil layer. Moreover, surface tension measurements confirmed that the emulsions were still in the miniemulsion regime and not in the microemulsion regime. The particle size can be tuned by controlling the duration during which the miniemulsion stayed in contact with the hexadecane layer, the interfacial area between the miniemulsion and the hexadecane layer and by the concentration of surfactant. Our method was applied to reduce the size of polystyrene and poly(methyl methacrylate) nanoparticles, nanocapsules of a copolymer of styrene and methyl methacrylic acid, and silica nanocapsules. This work demonstrated that a successful reduction of nanoparticle size in the miniemulsion process can be achieved without using excess amounts of surfactant. The method relies on building osmotic pressure in oil droplets dispersed in water which acts as semipermeable membrane.

Advanced density-based methods for the characterization of materials, binding events, and kinetics.

Investigations of the densities of chemicals and materials bring valuable insights into the fundamental understanding of matter and processes. Recently, advanced density-based methods have been developed with wide measurement ranges (i.e. 0-23 g cm-3), high resolutions (i.e. 10-6 g cm-3), compatibility with different types of samples and the requirement of extremely low volumes of sample (as low as a single cell). Certain methods, such as magnetic levitation, are inexpensive, portable and user-friendly. Advanced density-based methods are, therefore, beneficially used to obtain absolute density values, composition of mixtures, characteristics of binding events, and kinetics of chemical and biological processes. Herein, the principles and applications of magnetic levitation, acoustic levitation, electrodynamic balance, aqueous multiphase systems, and suspended microchannel resonators for materials science are discussed.

Zonated leucine sensing by Sestrin-mTORC1 in the liver controls the response to dietary leucine.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase controls growth in response to nutrients, including the amino acid leucine. In cultured cells, mTORC1 senses leucine through the leucine-binding Sestrin proteins, but the physiological functions and distribution of Sestrin-mediated leucine sensing in mammals are unknown. We find that mice lacking Sestrin1 and Sestrin2 cannot inhibit mTORC1 upon dietary leucine deprivation and suffer a rapid loss of white adipose tissue (WAT) and muscle. The WAT loss is driven by aberrant mTORC1 activity and fibroblast growth factor 21 (FGF21) production in the liver. Sestrin expression in the liver lobule is zonated, accounting for zone-specific regulation of mTORC1 activity and FGF21 induction by leucine. These results establish the mammalian Sestrins as physiological leucine sensors and reveal a spatial organization to nutrient sensing by the mTORC1 pathway.

Comparative Analysis of Mitochondrial Genomes in Gryllidea (Insecta: Orthoptera): Implications for Adaptive Evolution in Ant-Loving Crickets.

Species of infraorder Gryllidea, or crickets, are useful invertebrate models for studying developmental biology and neuroscience. They have also attracted attention as alternative protein sources for human food and animal feed. Mitochondrial genomic information on related invertebrates, such as katydids, and locusts, has recently become available in attempt to clarify the controversial classification schemes, although robust phylogenetic relationships with emphasis on crickets remain elusive. Here, we report newly sequenced complete mitochondrial genomes of crickets to study their phylogeny, genomic rearrangements, and adaptive evolution. First, we conducted de novo assembly of mitochondrial genomes from eight cricket species and annotated protein-coding genes and transfer and ribosomal RNAs using automatic annotations and manual curation. Next, by combining newly described protein-coding genes with public data of the complete Gryllidea genomes and gene annotations, we performed phylogenetic analysis and found gene order rearrangements in several branches. We further analyzed genetic signatures of selection in ant-loving crickets (Myrmecophilidae), which are small wingless crickets that inhabit ant nests. Three distinct approaches revealed two positively selected sites in the cox1 gene in these crickets. Protein 3D structural analyses suggested that these selected sites could influence the interaction of respiratory complex proteins, conferring benefits to ant-loving crickets with a unique ecological niche and morphology. These findings enhance our understanding of the genetic basis of cricket evolution without relying on estimates based on a limited number of molecular markers.

Constructing Adult Zebrafish Einthoven's Triangle to Define Electrical Heart Axes.

Zebrafish is a popular high-throughput vertebrate model to study human cardiac electrophysiology, arrhythmias, and myopathies. One reason for this popularity is the purported striking similarities between zebrafish and human electrocardiograms (ECGs). However, zebrafish electrical heart axes were unknown. It is impossible to define heart axis based on single-lead ECG because determination of an electrical heart axis in the frontal plane requires the use of the hexaxial reference system (or Cabrera system) derived from Einthoven's triangle. Construction of Einthoven's triangle requires simultaneous ECG recording from at least two Einthoven bipolar leads. Therefore, we systematically constructed the first zebrafish Einthoven's triangle by simultaneous bipolar dual-lead ECG recording to determine for the first time the three frontal electrical heart axes using the Cabrera system. Comparing zebrafish with human Einthoven's triangle reveals that their normal frontal electrical axes were reflections of each other across 0° in the Cabrera system. The responsible mechanisms involve zebrafish vs. human cardiac activation propagating in the same direction along the heart horizontal axis but in opposite directions along the heart longitudinal axis. The same observations are true for zebrafish vs. human cardiac repolarization. This study marks a technical breakthrough in the first bipolar dual-lead ECG recording in live adult zebrafish to construct for the first time zebrafish Einthoven's triangle. This first systematic analysis of the actual differences and similarities between normal adult zebrafish and human Einthoven's triangles unmasked differences and similarities in the underlying cardiac axis mechanisms. Insights of the live adult zebrafish main heart axis and its three frontal electrical heart axes provide critical contextual framework to interpret the clinical relevance of the adult zebrafish heart as model for human cardiac electrophysiology.

Ultrasmall Nanocapsules Obtained by Controlling Ostwald Ripening.

We describe here a method to synthesize ultrasmall nanocapsules with a diameter of 6 nm, exhibiting a well-defined core-shell morphology. Remarkably, the nanocapules are synthesized in a miniemulsion process without the need of large amounts of surfactant as commonly used in the microemulsion process. Ultrasmall nanocapsules with an oil core and a silica shell are formed by the concurrent processes of a sol-gel reaction and Ostwald ripening. Using solvents with different water solubilities and alkoxysilanes with different reactivities, we demonstrate that sizes of obtained nanocapsules depend on the ripening rate and alkoxysilane conversion rate. The method can be also used for encapsulating natural oils such as peppermint oil and limonene. This work shows that the Ostwald ripening phenomenon can be employed beneficially for the preparation of very small colloids.

Adult zebrafish ventricular electrical gradients as tissue mechanisms of ECG patterns under baseline vs. oxidative stress.

AIMS: In mammalian ventricles, electrical gradients establish electrical heterogeneities as essential tissue mechanisms to optimize mechanical efficiency and safeguard electrical stability. Electrical gradients shape mammalian electrocardiographic patterns; disturbance of electrical gradients is proarrhythmic. The zebrafish heart is a popular surrogate model for human cardiac electrophysiology thanks to its remarkable recapitulation of human electrocardiogram and ventricular action potential features. Yet, zebrafish ventricular electrical gradients are largely unexplored. The goal of this study is to define the zebrafish ventricular electrical gradients that shape the QRS complex and T wave patterns at baseline and under oxidative stress. METHODS AND RESULTS: We performed in vivo electrocardiography and ex vivo voltage-sensitive fluorescent epicardial and transmural optical mapping of adult zebrafish hearts at baseline and during acute H2O2 exposure. At baseline, apicobasal activation and basoapical repolarization gradients accounted for the polarity concordance between the QRS complex and T wave. During H2O2 exposure, differential regional impairment of activation and repolarization at the apex and base disrupted prior to baseline electrical gradients, resulting in either reversal or loss of polarity concordance between the QRS complex and T wave. KN-93, a specific calcium/calmodulin-dependent protein kinase II inhibitor (CaMKII), protected zebrafish hearts from H2O2 disruption of electrical gradients. The protection was complete if administered prior to oxidative stress exposure. CONCLUSIONS: Despite remarkable apparent similarities, zebrafish and human ventricular electrocardiographic patterns are mirror images supported by opposite electrical gradients. Like mammalian ventricles, zebrafish ventricles are also susceptible to H2O2 proarrhythmic perturbation via CaMKII activation. Our findings suggest that the adult zebrafish heart may constitute a clinically relevant model to investigate ventricular arrhythmias induced by oxidative stress. However, the fundamental ventricular activation and repolarization differences between the two species that we demonstrated in this study highlight the potential limitations when extrapolating results from zebrafish experiments to human cardiac electrophysiology, arrhythmias, and drug toxicities.

Revisiting Antiarrhythmic Drug Therapy for Atrial Fibrillation: Reviewing Lessons Learned and Redefining Therapeutic Paradigms.

Since the clinical use of digitalis as the first pharmacological therapy for atrial fibrillation (AF) 235 years ago in 1785, antiarrhythmic drug therapy has advanced considerably and become a cornerstone of AF clinical management. Yet, a preventive or curative panacea for sustained AF does not exist despite the rise of AF global prevalence to epidemiological proportions. While multiple elevated risk factors for AF have been established, the natural history and etiology of AF remain incompletely understood. In the present article, the first section selectively highlights some disappointing shortcomings and current efforts in antiarrhythmic drug therapy to uncover reasons why AF is such a clinical challenge. The second section discusses some modern takes on the natural history of AF as a relentless, progressive fibro-inflammatory "atriomyopathy." The final section emphasizes the need to redefine therapeutic strategies on par with new insights of AF pathophysiology.

Pre-exposure Prophylaxis in HIV Research: A Latent Dirichlet Allocation Analysis (GAPRESEARCH).

Pre-exposure prophylaxis (PrEP) has been shown to be an effective approach to prevent human immunodeficiency virus (HIV) infections; however, implementation of the service remains challenging. This global bibliometric analysis aims to describe the current trends in HIV research prevention through PrEP to reveal the potential gaps of knowledge and to put forward recommendations for future research. A bibliometric analysis was conducted through Web of Science from 1990 to 2017. Exploratory factor analysis was also employed to find research domains emerging from the abstracts' contents. Latent Dirichlet allocation, which is a topic modeling algorithm, was utilized to perform text mining and determine relationships among text documents. A total of 4852 papers regarding HIV PrEP research were retrieved. The number of papers and their impact has significantly increased. Preventing sexual transmissions, improving access, and quality of health-care services for current users, as well as men who have sex with men, pregnant women and children, were the research domains most related to PrEP. We found a data gap in research regarding sex workers, potential side effects of PrEP, and misjudgment toward PrEP users. Despite the growth in research about HIV PrEP, there exist barriers to scaling up the implementation of PrEP worldwide and for such intervention to reach its fWull potential. International research collaboration efforts to investigate the potential safety concerns of PrEP and develop strategies to eliminate social misjudgment against PrEP users are warranted. Addressing these knowledge gaps might facilitate the development of effective global implementation strategies for PrEP in the future.

Glypican-3-Specific CAR T Cells Coexpressing IL15 and IL21 Have Superior Expansion and Antitumor Activity against Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-related death in the world, and curative systemic therapies are lacking. Chimeric antigen receptor (CAR)-expressing T cells induce robust antitumor responses in patients with hematologic malignancies but have limited efficacy in patients with solid tumors, including HCC. IL15 and IL21 promote T-cell expansion, survival, and function and can improve the antitumor properties of T cells. We explored whether transgenic expression of IL15 and/or IL21 enhanced glypican-3-CAR (GPC3-CAR) T cells' antitumor properties against HCC. We previously optimized the costimulation in GPC3-CARs and selected a second-generation GPC3-CAR incorporating a 4-1BB costimulatory endodomain (GBBz) for development. Here, we generated constructs encoding IL15, IL21, or both with GBBz (15.GBBz, 21.GBBz, and 21.15.GBBz, respectively) and examined the ability of transduced T cells to kill, produce effector cytokines, and expand in an antigen-dependent manner. We performed gene-expression and phenotypic analyses of GPC3-CAR T cells and CRISPR-Cas9 knockout of the TCF7 gene. Finally, we measured GPC3-CAR T-cell antitumor activity in murine xenograft models of GPC3+ tumors. The increased proliferation of 21.15.GBBz T cells was at least in part dependent on the upregulation and maintenance of TCF-1 (encoded by TCF7) and associated with a higher percentage of stem cell memory and central memory populations after manufacturing. T cells expressing 21.15.GBBz had superior in vitro and in vivo expansion and persistence, and the most robust antitumor activity in vivo These results provided preclinical evidence to support the clinical evaluation of 21.15.GPC3-CAR T cells in patients with HCC.

Proarrhythmic Electrical Remodeling by Noncardiomyocytes at Interfaces With Cardiomyocytes Under Oxidative Stress.

Life-threatening ventricular arrhythmias, typically arising from interfaces between fibrosis and surviving cardiomyocytes, are feared sequelae of structurally remodeled hearts under oxidative stress. Incomplete understanding of the proarrhythmic electrical remodeling by fibrosis limits the development of novel antiarrhythmic strategies. To define the mechanistic determinants of the proarrhythmia in electrical crosstalk between cardiomyocytes and noncardiomyocytes, we developed a novel in vitro model of interface between neonatal rat ventricular cardiomyocytes (NRVMs) and controls [NRVMs or connexin43 (Cx43)-deficient HeLa cells] vs. Cx43+ noncardiomyocytes [aged rat ventricular myofibroblasts (ARVFs) or HeLaCx43 cells]. We performed high-speed voltage-sensitive optical imaging at baseline and following acute H2O2 exposure. In NRVM-NRVM and NRVM-HeLa controls, no arrhythmias occurred under either experimental condition. In the NRVM-ARVF and NRVM-HeLaCx43 groups, Cx43+ noncardiomyocytes enabled passive decremental propagation of electrical impulses and impaired NRVM activation and repolarization, thereby slowing conduction and prolonging action potential duration. Following H2O2 exposure, arrhythmia triggers, automaticity, and non-reentrant and reentrant arrhythmias emerged. This study reveals that myofibroblasts (which generate cardiac fibrosis) and other noncardiomyocytes can induce not only structural remodeling but also electrical remodeling and that electrical remodeling by noncardiomyocytes can be particularly arrhythmogenic in the presence of an oxidative burst. Synergistic electrical remodeling between H2O2 and noncardiomyocytes may account for the clinical arrhythmogenicity of myofibroblasts at fibrotic interfaces with cardiomyocytes in ischemic/non-ischemic cardiomyopathies. Understanding the enhanced arrhythmogenicity of synergistic electrical remodeling by H2O2 and noncardiomyocytes may guide novel safe-by-design antiarrhythmic strategies for next-generation iatrogenic interfaces between surviving native cardiomyocytes and exogenous stem cells or engineered tissues in cardiac regenerative therapies.

In Vivo Surface Electrocardiography for Adult Zebrafish.

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Regulating Payload Release from Hybrid Nanocapsules with Dual Silica/Polycaprolactone Shells.

We describe a facile strategy to synthesize hybrid nanocapsules with an oil core for hindering interactions between payloads and silica shell. Polycaprolactone/silica nanocapsules are synthesized by an interfacial sol-gel process occurring simultaneously with internal phase separation of the polymer produced by a miniemulsion-solvent evaporation technique. The localization of the polycaprolactone in the nanocapsules is depending on the ratio between polymer and silica. Formation of hybrid nanocapsules is found to significantly hinder interactions of drugs such as ibuprofen and carbamazepine with the silica surface.

Failure of Indomethacin and Radiation to Prevent Blast-induced Heterotopic Ossification in a Sprague-Dawley Rat Model.

BACKGROUND: Although use of nonsteroidal antiinflammatory drugs and low-dose irradiation has demonstrated efficacy in preventing heterotopic ossification (HO) after THA and surgical treatment of acetabular fractures, these modalities have not been assessed after traumatic blast amputations where HO is a common complication that can arise in the residual limb. QUESTIONS/PURPOSES: The purpose of this study was to investigate the effectiveness of indomethacin and irradiation in preventing HO induced by high-energy blast trauma in a rat model. METHODS: Thirty-six Sprague-Dawley rats underwent hind limb blast amputation with a submerged explosive under water followed by irrigation and primary wound closure. One group (n = 12) received oral indomethacin for 10 days starting on postoperative Day 1. Another group (n = 12) received a single dose of 8 Gy irradiation to the residual limb on postoperative Day 3. A control group (n = 12) did not receive either. Wound healing and clinical course were monitored in all animals until euthanasia at 24 weeks. Serial radiographs were taken immediately postoperatively, at 10 days, and every 4 weeks thereafter to monitor the time course of ectopic bone formation until euthanasia. Five independent graders evaluated the 24-week radiographs to quantitatively assess severity and qualitatively assess the pattern of HO using a modified Potter scale from 0 to 3. Assessment of grading reproducibility yielded a Fleiss statistic of 0.41 and 0.37 for severity and type, respectively. By extrapolation from human clinical trials, a minimum clinically important difference in HO severity was empirically determined to be two full grades or progression of absolute grade to the most severe. RESULTS: We found no differences in mean HO severity scores among the three study groups (indomethacin 0.90 ± 0.46 [95% confidence interval {CI}, 0.60-1.19]; radiation 1.34 ± 0.59 [95% CI, 0.95-1.74]; control 0.95 ± 0.55 [95% CI, 0.60-1.30]; p = 0.100). For qualitative HO type scores, the radiation group had a higher HO type than both indomethacin and controls, but indomethacin was no different than controls (indomethacin 1.08 ± 0.66 [95% CI, 0.67-1.50]; radiation 1.89 ± 0.76 [95% CI, 1.38-2.40]; control 1.10 ± 0.62 [95% CI, 0.70-1.50]; p = 0.013). The lower bound of the 95% CI on mean severity in the indomethacin group and the upper bound of the radiation group barely spanned a full grade and involved only numeric grades < 2, suggesting that even if a small difference in severity could be detected, it would be less than our a priori-defined minimum clinically important difference and any differences that might be present are unlikely to be clinically meaningful. CONCLUSIONS: This work unexpectedly demonstrated that, compared with controls, indomethacin and irradiation provide no effective prophylaxis against HO in the residual limb after high-energy blast amputation in a rat model. Such an observation is contrary to the civilian experience and may be potentially explained by either a different pathogenesis for blast-induced HO or a stimulus that overwhelms conventional regimens used to prevent HO in the civilian population. CLINICAL RELEVANCE: HO in the residual limb after high-energy traumatic blast amputation will likely require novel approaches for prevention and management.

Hybrid density functional study on the electronic structures and properties of P3HT-PbS and P3HT-CdS hybrid interface for photovoltaic applications.

The efficiency of charge transport mainly depends on the interfacial energy level alignment between the conjugated polymer and the inorganic substrate. It provides an accurate understanding, predicting as well as controlling the optimal power conversion efficiency of various type of hybrid photovoltaic systems. In this article, we use hybrid functional (HSE06) to study the electronic structures and properties at the interface of poly(3-hexylthiophene)(P3HT)/CdS and P3HT/PbS for solar cell applications. We found that the dangling bonds at the inorganic surface introduce in-gap states and greatly reduce the device performance. We used pseudo-hydrogen atoms as the passivation agent to remove the dangling bonds and eliminate the in-gap states to construct the energy alignment at the hybrid interface. The calculated interfacial density of states reveal a better performance in P3HT/CdS, compared to P3HT/PbS. P3HT/CdS possesses a LUMOP3HT /CBMCdS and HOMOP3HT /VBMCdS energy offset large enough for sufficient exciton separation across the interface and prevents charge recombination. In contrast, the reason for low power conversion efficiency in P3HT/PbS lies on its HOMOP3HT /VBMPbS offset which is too small to break the exciton binding energy for charge separation. Moreover, we reported the dependency of the energy level alignment and open circuit voltage on the interfacial molecular orientations. Our DFT calculation can be used to predict candidate materials for the development of efficiency optoelectronic devices. © 2018 Wiley Periodicals, Inc.

Remarkable charge-transfer mobility from [6] to [10]phenacene as a high performance p-type organic semiconductor.

The relationship between structure and charge transport properties of phenacene organic semiconductors has been studied with focus on [6] → [10]phenacene. Upon inserting phenyl rings, the π-extended structure results in strong electronic coupling interactions and reduction of reorganization energy. Using the classical Marcus charge transport theory, we predict that hole mobility in the phenacene series increases gradually up to 8.0 cm2 V-1 s-1 at [10]phenacene. This is remarkably high among other discovered OSCs, surpassing that of pentacene. Moreover, we notice that the experimental hole mobility of [6]phenacene is unusually low, inconsistent with other members in the same series. Thus, we performed full structural relaxation on phenacene and revealed similarities between theoretical and experimental crystal structures for all the members except [6]phenacene. We propose a new structure of [6]phenacene under the consideration of van der Waals force with smaller lattice parameters a* and b* compared to the experimental structure. Our new structural calculation fits well with the existing trend of hole mobility, energy gaps, effective masses, bandwidth and lattice parameters. Single-shot G0W0 calculations are performed to verify our structures. The results give a hint that the improvement in [6]phenacene efficiency lies on the intermolecular distance along the stacking direction of the crystal. Phenacene compounds generally have small effective masses, high charge transfer integrals and moderate reorganization energies necessary for hole transport. Our results suggest that the phenacene series, in particular [6] → [10]phenacene, have high charge mobility and air stability essential for achieving high efficiency electronic devices.

Integrating light-sheet imaging with virtual reality to recapitulate developmental cardiac mechanics.

Currently, there is a limited ability to interactively study developmental cardiac mechanics and physiology. We therefore combined light-sheet fluorescence microscopy (LSFM) with virtual reality (VR) to provide a hybrid platform for 3D architecture and time-dependent cardiac contractile function characterization. By taking advantage of the rapid acquisition, high axial resolution, low phototoxicity, and high fidelity in 3D and 4D (3D spatial + 1D time or spectra), this VR-LSFM hybrid methodology enables interactive visualization and quantification otherwise not available by conventional methods, such as routine optical microscopes. We hereby demonstrate multiscale applicability of VR-LSFM to (a) interrogate skin fibroblasts interacting with a hyaluronic acid-based hydrogel, (b) navigate through the endocardial trabecular network during zebrafish development, and (c) localize gene therapy-mediated potassium channel expression in adult murine hearts. We further combined our batch intensity normalized segmentation algorithm with deformable image registration to interface a VR environment with imaging computation for the analysis of cardiac contraction. Thus, the VR-LSFM hybrid platform demonstrates an efficient and robust framework for creating a user-directed microenvironment in which we uncovered developmental cardiac mechanics and physiology with high spatiotemporal resolution.

Pulsatile Lavage of Musculoskeletal Wounds Causes Muscle Necrosis and Dystrophic Calcification in a Rat Model.

BACKGROUND: Adequate irrigation of open musculoskeletal injuries is considered the standard of care to decrease bacterial load and other contaminants. While the benefit of debris removal compared with the risk of further seeding by high-pressure lavage has been studied, the effects of irrigation on muscle have been infrequently reported. Our aim in the present study was to assess relative damage to muscle by pulsatile lavage compared with bulb-syringe irrigation. METHODS: In an animal model of heterotopic ossification, 24 Sprague-Dawley rats underwent hindlimb blast amputation via detonation of a submerged explosive, with subsequent through-the-knee surgical amputation proximal to the zone of injury. All wounds were irrigated and underwent primary closure. In 12 of the animals, pulsatile lavage (20 psi [138 kPa]) was used as the irrigation method, and in the other 12 animals, bulb-syringe irrigation was performed. A third group of 6 rats did not undergo the blast procedure but instead underwent surgical incision into the left thigh muscle followed by pulsatile lavage. Serial radiographs of the animals were made to monitor the formation of soft-tissue radiopaque lesions until euthanasia at 6 months. Image-guided muscle biopsies were performed at 8 weeks and 6 months (at euthanasia) on representative animals from each group. Histological analysis was performed with hematoxylin and eosin, alizarin red, and von Kossa staining on interval biopsy and postmortem specimens. RESULTS: All animals managed with pulsatile lavage, with or without blast injury, developed soft-tissue radiopaque lesions, whereas no animal that had bulb-syringe irrigation developed these lesions (p = 0.001). Five of the 12 animals that underwent blast amputation with pulsatile lavage experienced wound complications, whereas no animal in the other 2 groups experienced wound complications (p = 0.014). Radiopaque lesions appeared approximately 10 days postoperatively, increased in density until approximately 16 weeks, then demonstrated signs of variable regression. Histological analysis of interval biopsy and postmortem specimens demonstrated tissue damage with inflammatory cells, cell death, and dystrophic calcification. CONCLUSIONS: Pulsatile lavage of musculoskeletal wounds can cause irreversible insult to tissue, resulting in myonecrosis and dystrophic calcification. CLINICAL RELEVANCE: The benefits and offsetting harm of pulsatile lavage (20 psi) should be considered before its routine use in the management of musculoskeletal wounds.