Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.

Epicardial deletion of Sox9 leads to myxomatous valve degeneration and identifies Cd109 as a novel gene associated with valve development.

Epicardial-derived cells (EPDCs) are involved in the regulation of myocardial growth and coronary vascularization and are critically important for proper development of the atrioventricular (AV) valves. SOX9 is a transcription factor expressed in a variety of epithelial and mesenchymal cells in the developing heart, including EPDCs. To determine the role of SOX9 in epicardial development, an epicardial-specific Sox9 knockout mouse model was generated. Deleting Sox9 from the epicardial cell lineage impairs the ability of EPDCs to invade both the ventricular myocardium and the developing AV valves. After birth, the mitral valves of these mice become myxomatous with associated abnormalities in extracellular matrix organization. This phenotype is reminiscent of that seen in humans with myxomatous mitral valve disease (MVD). An RNA-seq analysis was conducted in an effort to identify genes associated with this myxomatous degeneration. From this experiment, Cd109 was identified as a gene associated with myxomatous valve pathogenesis in this model. Cd109 has never been described in the context of heart development or valve disease. This study highlights the importance of SOX9 in the regulation of epicardial cell invasion-emphasizing the importance of EPDCs in regulating AV valve development and homeostasis-and reports a novel expression profile of Cd109, a gene with previously unknown relevance in heart development.

A Mobile App for Postoperative Pain Management Among Older Veterans Undergoing Total Knee Arthroplasty: Mixed Methods Feasibility and Acceptability Pilot Study.

BACKGROUND: Prescription opioid misuse risk is disproportionate among veterans; military veterans wounded in combat misuse prescription opioids at an even higher rate (46.2%). Opioid misuse is costly in terms of morbidity, mortality, and humanitarian and economic burden and costs the Civilian Health and Medical Program of the Department of Veterans Affairs more than US $1.13 billion annually. Preventing opioid misuse at the time of prescription is a critical component in the response to the opioid crisis. The CPMRx mobile app has been shown to decrease the odds of opioid misuse during the postoperative period. OBJECTIVE: The overarching purpose of this feasibility pilot study was to explore whether deploying a mobile app (CPMRx) to track postoperative pain and medication use is feasible in a Department of Veterans Affairs medical center. In support of this goal, we had four complementary specific aims: (1) determine the technological and logistical feasibility of the mobile app, (2) assess the acceptability of the mobile app to participants, (3) measure demand for and engagement with the mobile app, and (4) explore the potential use of the mobile app to patients and providers. METHODS: Participants (N=10) were veterans undergoing total knee arthroplasty within the Veterans Health Administration provided with the CPMRx app to self-manage their pain during their 7-day at-home recovery following surgery. CPMRx uses scientifically validated tools to help clinicians understand how a patient can use the least amount of medication while getting the most benefit. The suite of software includes a mobile app for patients that includes a behavioral health intervention and a clinical decision support tool for health care providers that provides feedback about pain and medication use trends. Patients filled out paper questionnaires regarding acceptability at their postoperative follow-up appointment. RESULTS: Overall, quantitative measures of acceptability were high. The average rating for the amount of time required to use the app was 4.9 of 5 (5="very little"), and the average rating for ease of use was 4.4 of 5 (5="very easy"). Open-ended questions also revealed that most participants found ease of use to be high. Demand and engagement were high as well with a mean number of mobile app entries of 34.1 (SD 20.1) during the postoperative period. There were no reported technological or logistical issues with the mobile app. Participants took an average of 25.13 (SD 14.37) opioid tablets to manage their postoperative pain. CONCLUSIONS: Results of this study revealed that the use of a mobile app for pain and medication management during postoperative recovery was both feasible and acceptable in older veterans undergoing total knee arthroplasty within the Veterans Health Administration. The wide variation in opioid consumption across participants revealed the potential use of the mobile app to provide actionable insights to clinicians if adopted more widely.

Local blockade of tacrolimus promotes T-cell-mediated tumor regression in systemically immunosuppressed hosts.

BACKGROUND: Immunosuppressive drugs such as tacrolimus have revolutionized our ability to transplant organs between individuals. Tacrolimus acts systemically to suppress the activity of T-cells within and around transplanted organs. However, tacrolimus also suppresses T-cell function in the skin, contributing to a high incidence of skin cancer and associated mortality and morbidity in solid organ transplant recipients. Here, we aimed to identify a compound capable of re-establishing antitumor T-cell control in the skin despite the presence of tacrolimus. METHODS: In this study, we performed time-resolved fluorescence resonance energy transfer to identify molecules capable of antagonizing the interaction between tacrolimus and FKBP12. The capacity of these molecules to rescue mouse and human T-cell function in the presence of tacrolimus was determined in vitro, and the antitumor effect of the lead compound, Q-2361, was assessed in "regressor" models of skin cancer in immunosuppressed mice. Systemic CD8 T-cell depletion and analyses of intratumoral T-cell activation markers and effector molecule production were performed to determine the mechanism of tumor rejection. Pharmacokinetic studies of topically applied Q-2361 were performed to assess skin and systemic drug exposure. RESULTS: Q-2361 potently blocked the interaction between tacrolimus and FKBP12 and reversed the inhibition of the nuclear factor of activated T cells activation by tacrolimus following T-cell receptor engagement in human Jurkat cells. Q-2361 rescued T-cell function in the presence of tacrolimus, rapamycin, and everolimus. Intratumoral injection of Q-2361-induced tumor regression in mice systemically immune suppressed with tacrolimus. Mechanistically, Q-2361 treatment permitted T-cell activation, proliferation, and effector function within tumors. When CD8 T cells were depleted, Q-2361 could not induce tumor regression. A simple solution-based Q-2361 topical formulation achieved high and sustained residence in the skin with negligible drug in the blood. CONCLUSIONS: Our findings demonstrate that the local application of Q-2361 permits T-cells to become activated driving tumor rejection in the presence of tacrolimus. The data presented here suggests that topically applied Q-2361 has great potential for the reactivation of T-cells in the skin but not systemically, and therefore represents a promising strategy to prevent or treat skin malignancies in immunosuppressed organ transplant recipients.

Grambank reveals the importance of genealogical constraints on linguistic diversity and highlights the impact of language loss.

While global patterns of human genetic diversity are increasingly well characterized, the diversity of human languages remains less systematically described. Here, we outline the Grambank database. With over 400,000 data points and 2400 languages, Grambank is the largest comparative grammatical database available. The comprehensiveness of Grambank allows us to quantify the relative effects of genealogical inheritance and geographic proximity on the structural diversity of the world's languages, evaluate constraints on linguistic diversity, and identify the world's most unusual languages. An analysis of the consequences of language loss reveals that the reduction in diversity will be strikingly uneven across the major linguistic regions of the world. Without sustained efforts to document and revitalize endangered languages, our linguistic window into human history, cognition, and culture will be seriously fragmented.

Analysis of the U.S. Military Trauma System in Accordance With Doctrinal Levels of Warfare.

INTRODUCTION: In recent conflicts, the Joint Theater Trauma System (JTTS) led the systematic approach to improve battlefield trauma care, substantially contributing to the unprecedented survival of combat casualties. The Joint Trauma System (JTS) was codified in 2016 to preserve the lessons learned and functions of the JTTS, including the Department of Defense Trauma Registry. Concurrently, Combatant Commands (CCMD) were directed to establish CCMD Trauma Systems (CTS) "modeled after the JTTS" and to maintain a baseline of core functions intended to rapidly scale as needed. The complex nature of both CCMDs and the military trauma system has challenged the full implementation of the CTS. Analyzing the historical experiences of the JTTS, JTS, and CTS within a military doctrinal framework might enable the further success of the military trauma system. METHODS: The strategic, operational, and tactical levels of warfare, in accordance with Joint Publication 1-0, Doctrine of the Armed Forces of the United States, and Joint Publication 3-0, Joint Operations, established the analytic framework for this study. The literature regarding the JTTS, CTS, and JTS was reviewed for relevant information concerning organizational structure and functions of trauma system performance improvement (PI) capabilities. A comprehensive analysis was performed using a thematic approach to evaluating descriptive data contained within the collected data set. Deployed trauma system PI tasks, functions, and responsibilities were identified, defined, and correlated according to the respective levels of warfare. RESULTS: The comprehensive analysis revealed both discrete and overlapping tasks, functions, and responsibilities of the trauma system PI capabilities at each of the three levels of warfare. Strategic-level actions were categorized according to 12 distinct themes: reduce mortality; strategic reporting; centralized trauma registry; strategic communications; centralized organization; direct support to CCMDs; Department of Defense policy and doctrine; strategic-level PI; clinical practice guidelines; training and readiness standards; force structure, standardization, and interoperability; and research and development. Operational-level actions were categorized according to seven distinct themes: theater trauma system policies and requirements; theater trauma system leadership; stakeholder coordination; theater communication; theater standards for readiness and skill sustainment; trauma system planning; and medical logistics support. Tactical-level actions were categorized according to seven distinct themes: trauma system personnel; PI; documentation enforcement and patient care data collection; tactical planning recommendations for employing medical assets; research support; communication and reporting; and training and skills sustainment. CONCLUSION: The deployed U.S. military trauma system requires a robust PI capability to optimize combat casualty care. Policy updates, a joint military trauma system doctrine, and force design updates are necessary for deployed military trauma system PI capabilities to function optimally across all levels of warfare.

Injury, illness, and emotion: A review of the motivational continuum from trauma through recovery from an ecological perspective.

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Co-designing a curriculum model for career education: perspectives from regional communities in Australia.

The vocational experiences and skills of young adolescents could be infused into formal education by identifying career competencies to be taught within the academic curriculum. Such curriculum practices that embed educational and career pathways must also include the perspectives of students and the community, particularly those from marginalised groups. Drawing on data from 111 teachers, principals, carers and students, this paper presents research undertaken to co-design career education lesson plans within an infused model of the curriculum for early Middle Year students from regional, rural, and remote Australia. The lesson plans and activities were designed to allow for meaningful self-reflection and goal-setting that could be seamlessly infused into the formal curriculum and help embed early-stage career education. The paper concludes by projecting opportunities and challenges for seamless curriculum integration, while pertinent to the Australian context, can also be read with broader relevance to other educational systems and schools.

Changes in Oxygenation Levels During Moderate Altitude Simulation (Hypoxia-Induced): A Pilot Study Investigating the Impact of Skin Pigmentation in Pulse Oximetry.

The current COVID-19 pandemic has shown us that the pulse oximeter is a key medical device for monitoring blood-oxygen levels non-invasively in patients with chronic or acute illness. It has also emphasised limitations in accuracy for individuals with darker skin pigmentation, calling for new methods to provide better measurements. The aim of our study is to identify the impact of skin pigmentation on pulse oximeter measurements. We also explored the benefits of a multi-wavelength approach with an induced change of arterial oxygen saturation. A total of 20 healthy volunteers were recruited. We used time domain diffuse reflectance spectroscopy (TDDRS) from a broad band light source, collecting spectra from the index finger along with three different pulse oximeters used simultaneously for monitoring purposes. Five acute hypoxic events were induced by administering 11% FiO2, produced by a Hypoxico altitude training system, for 120 sec through a face mask with a one-way valve. Our multi-wavelength approach revealed a correlation between the signature of skin pigmentation and the dynamic range of oxygen saturation measurements. Principal component analysis (PCA) showed separation between a range of different pigmented volunteers (PC1 = 56.00%) and oxygen saturation (PC2 = 22.99%). This emphasises the need to take into account skin pigmentation in oximeter measurements. This preliminary study serves to validate the need to better understand the impact of skin pigmentation absorption on optical readings in pulse oximeters. Multi-wavelength approaches have the potential to enable robust and accurate measurements across diverse populations.

Sox9 Expression in the Second Heart Field; A Morphological Assessment of the Importance to Cardiac Development with Emphasis on Atrioventricular Septation.

Failure to form the septal structures that separate the left and right cardiac chambers results in defects that allow shunting of blood from one side of the heart to the other, leading to the mixing of oxygenated and de-oxygenated blood. The atrioventricular (AV) mesenchymal complex, consisting of the AV cushions, the Dorsal Mesenchymal Protrusion (DMP), and the mesenchymal cap, plays a crucial role in AV septation. Cells found in these structures derive from different cell lineages. In this study we have investigated the role of the transcription factor Sox9 in the Second Heart Field (SHF) with the emphasis on the formation of the atrioventricular septal complex. Using a mouse model in which Sox9 is conditionally deleted from the SHF we demonstrate that in this model virtually all mouse embryos develop septal abnormalities, including complete atrioventricular septal defects (cAVSDs) and isolated ventricular septal defects. Our morphological analyses indicate that perturbation of the development of the mesenchymal cap appears to play a crucial role in the pathogenesis of the atrial septal defects observed in the AVSDs and suggests that this component of the AV mesenchymal complex might play a more important role in AV septation than previously appreciated.

Cellular and Molecular Mechanisms of MEK1 Inhibitor-Induced Cardiotoxicity.

Background: Trametinib is a MEK1 (mitogen-activated extracellular signal-related kinase kinase 1) inhibitor used in the treatment of BRAF (rapid accelerated fibrosarcoma B-type)-mutated metastatic melanoma. Roughly 11% of patients develop cardiomyopathy following long-term trametinib exposure. Although described clinically, the molecular landscape of trametinib cardiotoxicity has not been characterized. Objectives: The aim of this study was to test the hypothesis that trametinib promotes widespread transcriptomic and cellular changes consistent with oxidative stress and impairs cardiac function. Methods: Mice were treated with trametinib (1 mg/kg/d). Echocardiography was performed pre- and post-treatment. Gross, histopathologic, and biochemical assessments were performed to probe for molecular and cellular changes. Human cardiac organoids were used as an in vitro measurement of cardiotoxicity and recovery. Results: Long-term administration of trametinib was associated with significant reductions in survival and left ventricular ejection fraction. Histologic analyses of the heart revealed myocardial vacuolization and calcification in 28% of animals. Bulk RNA sequencing identified 435 differentially expressed genes and 116 differential signaling pathways following trametinib treatment. Upstream gene analysis predicted interleukin-6 as a regulator of 17 relevant differentially expressed genes, suggestive of PI3K/AKT and JAK/STAT activation, which was subsequently validated. Trametinib hearts displayed elevated markers of oxidative stress, myofibrillar degeneration, an 11-fold down-regulation of the apelin receptor, and connexin-43 mislocalization. To confirm the direct cardiotoxic effects of trametinib, human cardiac organoids were treated for 6 days, followed by a 6-day media-only recovery. Trametinib-treated organoids exhibited reductions in diameter and contractility, followed by partial recovery with removal of treatment. Conclusions: These data describe pathologic changes observed in trametinib cardiotoxicity, supporting the exploration of drug holidays and alternative pharmacologic strategies for disease prevention.

Addressing phase-curvature in Fourier ptychography.

In Fourier ptychography, multiple low resolution images are captured and subsequently combined computationally into a high-resolution, large-field of view micrograph. A theoretical image-formation model based on the assumption of plane-wave illumination from various directions is commonly used, to stitch together the captured information into a high synthetic aperture. The underlying far-field (Fraunhofer) diffraction assumption connects the source, sample, and pupil planes by Fourier transforms. While computationally simple, this assumption neglects phase-curvature due to non-planar illumination from point sources as well as phase-curvature from finite-conjugate microscopes (e.g., using a single-lens for image-formation). We describe a simple, efficient, and accurate extension of Fourier ptychography by embedding the effect of phase-curvature into the underlying forward model. With the improved forward model proposed here, quantitative phase reconstruction is possible even for wide fields-of-views and without the need of image segmentation. Lastly, the proposed method is computationally efficient, requiring only two multiplications: prior and following the reconstruction.

Snapshot volumetric imaging with engineered point-spread functions.

The biological world involves intracellular and intercellular interactions that occur at high speed, at multiple scales and in three dimensions. Acquiring 3D images, however, typically requires a compromise in either spatial or temporal resolution compared to 2D imaging. Conventional 2D fluorescence imaging provides high spatial resolution but requires plane-by-plane imaging of volumes. Conversely, snapshot methods such as light-field microscopy allow video-rate imaging, but at the cost of spatial resolution. Here we introduce 3D engineered point-spread function microscopy (3D-EPM), enabling snapshot imaging of real-world 3D extended biological structures while retaining the native resolution of the microscope in space and time. Our new computational recovery strategy is the key to volumetrically reconstructing arbitrary 3D structures from the information encapsulated in 2D raw EPM images. We validate our technique on both point-like and extended samples, and demonstrate its power by imaging the intracellular motion of chloroplasts undergoing cyclosis in a sample of Egeria densa. Our technique represents a generalised computational methodology for 3D image recovery which is readily adapted to a diverse range of existing microscopy platforms and engineered point-spread functions. We therefore expect it to find broad applicability in the study of rapid biological dynamics in 3D.

High-speed multi-objective Fourier ptychographic microscopy.

The ability of a microscope to rapidly acquire wide-field, high-resolution images is limited by both the optical performance of the microscope objective and the bandwidth of the detector. The use of multiple detectors can increase electronic-acquisition bandwidth, but the use of multiple parallel objectives is problematic since phase coherence is required across the multiple apertures. We report a new synthetic-aperture microscopy technique based on Fourier ptychography, where both the illumination and image-space numerical apertures are synthesized, using a spherical array of low-power microscope objectives that focus images onto mutually incoherent detectors. Phase coherence across apertures is achieved by capturing diffracted fields during angular illumination and using ptychographic reconstruction to synthesize wide-field, high-resolution, amplitude and phase images. Compared to conventional Fourier ptychography, the use of multiple objectives reduces image acquisition times by increasing the area for sampling the diffracted field. We demonstrate the proposed scaleable architecture with a nine-objective microscope that generates an 89-megapixel, 1.1 µm resolution image nine-times faster than can be achieved with a single-objective Fourier-ptychographic microscope. New calibration procedures and reconstruction algorithms enable the use of low-cost 3D-printed components for longitudinal biological sample imaging. Our technique offers a route to high-speed, gigapixel microscopy, for example, imaging the dynamics of large numbers of cells at scales ranging from sub-micron to centimetre, with an enhanced possibility to capture rare phenomena.

Pixel super-resolution with spatially entangled photons.

Pixelation occurs in many imaging systems and limits the spatial resolution of the acquired images. This effect is notably present in quantum imaging experiments with correlated photons in which the number of pixels used to detect coincidences is often limited by the sensor technology or the acquisition speed. Here, we introduce a pixel super-resolution technique based on measuring the full spatially-resolved joint probability distribution (JPD) of spatially-entangled photons. Without shifting optical elements or using prior information, our technique increases the pixel resolution of the imaging system by a factor two and enables retrieval of spatial information lost due to undersampling. We demonstrate its use in various quantum imaging protocols using photon pairs, including quantum illumination, entanglement-enabled quantum holography, and in a full-field version of N00N-state quantum holography. The JPD pixel super-resolution technique can benefit any full-field imaging system limited by the sensor spatial resolution, including all already established and future photon-correlation-based quantum imaging schemes, bringing these techniques closer to real-world applications.

Uncovering how animals use combinations of magnetic field properties to navigate: a computational approach.

Certain animal species use the earth's magnetic field (i.e., magnetoreception) in conjunction with other sensory modalities to navigate long distances. It is hypothesized that several animals use combinations of magnetic inclination and intensity as unique signatures for localization, potentially enabling migration without a pre-surveyed map. However, it is unknown how animals use magnetic signatures to generate guidance commands. While animal experiments have been invaluable in advancing this area, it is a difficult phenomenon to study in vivo or in situ. Modeling and simulation present a powerful complementary tool that can be used to investigate whether and how animals use magnetic signatures to navigate. This perspective article summarizes work we have conducted that systematically and mechanistically uses modeling and simulation to study the use of magnetic signatures. We have studied this phenomenon from simulated agents that navigate in simple and abstract environments, to physical devices that navigate in realistic environments. The results have consistently demonstrated that this is a plausible way in which animals might navigate, and provided early insights into the environmental and animal-specific factors that are most important to this navigation strategy.

Integrated neuroimmune processing of threat, injury, and illness: An ecological framework mapping social alienation onto lifetime health vulnerability.

Social alienation is a pre-eminent ecological threat for humans. In clinical and social care settings its impact is acknowledged in conditions as diverse as severe mood disturbance, chronic pain, and metabolic non-communicable diseases. An integrated psychoneuroimmune perspective shows how threat, injury, healing, and recovery follow through as a continuous process, but accepted cultural and clinical paradigms separating mental from physical illness provide little common ground on which to analyse and apply this continuum in practice. By reviewing the ecological relationships between emotional threat, tissue dyshomeostasis and injury, infection, pain, and mood this article explores not only how primeval somatic responses underpin the evolutionary foundations of depression and somatisation, but also links them to escalating physical non-communicable disease through archived socioeconomic adversity (allostatic load). Social alienation (in the absence of trauma) may prime and activate this ancient repertoire in which sensitised responses lay the foundation for persistent maladaptive states of aversive sensory misinterpretation, behavioural avoidance, anhedonia, and neuroinflammation presenting as widespread non-nociceptive pain, non-pain somatisation, and severe depression. The ecological perspective illuminates perverse clinical presentations, shows how some approaches to care may facilitate self-reinforcement in maladaptive syndromes, and offers pointers for inclusive rehabilitative clinical and social care.

A farewell to R: time-series models for tracking and forecasting epidemics.

The time-dependent reproduction number, Rt, is a key metric used by epidemiologists to assess the current state of an outbreak of an infectious disease. This quantity is usually estimated using time-series observations on new infections combined with assumptions about the distribution of the serial interval of transmissions. Bayesian methods are often used with the new cases data smoothed using a simple, but to some extent arbitrary, moving average. This paper describes a new class of time-series models, estimated by classical statistical methods, for tracking and forecasting the growth rate of new cases and deaths. Very few assumptions are needed and those that are made can be tested. Estimates of Rt, together with their standard deviations, are obtained as a by-product.

Role of the Epicardium in the Development of the Atrioventricular Valves and Its Relevance to the Pathogenesis of Myxomatous Valve Disease.

This paper is dedicated to the memory of Dr. Adriana "Adri" Gittenberger-de Groot and in appreciation of her work in the field of developmental cardiovascular biology and the legacy that she has left behind. During her impressive career, Dr. Gittenberger-de Groot studied many aspects of heart development, including aspects of cardiac valve formation and disease and the role of the epicardium in the formation of the heart. In this contribution, we review some of the work on the role of epicardially-derived cells (EPDCs) in the development of the atrioventricular valves and their potential involvement in the pathogenesis of myxomatous valve disease (MVD). We provide an overview of critical events in the development of the atrioventricular junction, discuss the role of the epicardium in these events, and illustrate how interfering with molecular mechanisms that are involved in the epicardial-dependent formation of the atrioventricular junction leads to a number of abnormalities. These abnormalities include defects of the AV valves that resemble those observed in humans that suffer from MVD. The studies demonstrate the importance of the epicardium for the proper formation and maturation of the AV valves and show that the possibility of epicardial-associated developmental defects should be taken into consideration when determining the genetic origin and pathogenesis of MVD.

Modulating Cas9 activity for precision gene editing.

The CRISPR/Cas9 is a RNA-guided nuclease complex that can be specifically programmed to target a user-specified DNA sequence. It has been a powerful and effective tool of genome editing. However, off-target activity of the Cas9 nuclease limits its potential use in the correction of inherited diseases and bona fide gene editing. Various protein engineering and guide RNA selection strategies have been utilized to improve Cas9-based genome-editing specificity and efficiency. We, however, have not yet achieved a degree of safety such that Cas9 gene editing approaches could be applicable in clinical settings. Here, we discuss the recently developed and precise gene editing technologies based on spCas9. Furthermore, we describe Cas9 modulating tools to increase the fidelity of the CRISPR/Cas9 system. These studies suggest that there is still a need for pharmaceutical modulation of Cas9 activity during gene editing procedures. Pharmaceutical modulation of Cas9 nuclease activity at on-target or off-target genomic loci could 1 day allow researchers to develop robust and precise therapeutical strategies in gene editing.