Facial analytics based on a coordinate extrapolation system (zFACE) for morphometric phenotyping of developing zebrafish.

Facial development requires a complex and coordinated series of cellular events that, when perturbed, can lead to structural birth defects. A quantitative approach to quickly assess morphological changes could address how genetic or environmental inputs lead to differences in facial shape and promote malformations. Here, we report on a method to rapidly analyze craniofacial development in zebrafish embryos using facial analytics based on a coordinate extrapolation system, termed zFACE. Confocal images capture facial structures and morphometric data are quantified based on anatomical landmarks present during development. The quantitative morphometric data can detect phenotypic variation and inform on changes in facial morphology. We applied this approach to show that loss of smarca4a in developing zebrafish leads to craniofacial anomalies, microcephaly and alterations in brain morphology. These changes are characteristic of Coffin-Siris syndrome, a rare human genetic disorder associated with mutations in SMARCA4. Multivariate analysis of zFACE data facilitated the classification of smarca4a mutants based on changes in specific phenotypic characteristics. Together, zFACE provides a way to rapidly and quantitatively assess the impact of genetic alterations on craniofacial development in zebrafish.

A miniaturized 3D printed pressure regulator (µPR) for microfluidic cell culture applications.

Well-defined fluid flows are the hallmark feature of microfluidic culture systems and enable precise control over biophysical and biochemical cues at the cellular scale. Microfluidic flow control is generally achieved using displacement-based (e.g., syringe or peristaltic pumps) or pressure-controlled techniques that provide numerous perfusion options, including constant, ramped, and pulsed flows. However, it can be challenging to integrate these large form-factor devices and accompanying peripherals into incubators or other confined environments. In addition, microfluidic culture studies are primarily carried out under constant perfusion conditions and more complex flow capabilities are often unused. Thus, there is a need for a simplified flow control platform that provides standard perfusion capabilities and can be easily integrated into incubated environments. To this end, we introduce a tunable, 3D printed micro pressure regulator (µPR) and show that it can provide robust flow control capabilities when combined with a battery-powered miniature air pump to support microfluidic applications. We detail the design and fabrication of the µPR and: (i) demonstrate a tunable outlet pressure range relevant for microfluidic applications (1-10 kPa), (ii) highlight dynamic control capabilities in a microfluidic network, (iii) and maintain human umbilical vein endothelial cells (HUVECs) in a multi-compartment culture device under continuous perfusion conditions. We anticipate that our 3D printed fabrication approach and open-access designs will enable customized µPRs that can support a broad range of microfluidic applications.

Microengineered 3D Collagen Gels with Independently Tunable Fiber Anisotropy and Directionality.

Cellular processes, including differentiation, proliferation, and migration, have been linked to the alignment (anisotropy) and orientation (directionality) of collagen fibers in the native extracellular matrix (ECM). Given the critical role that biophysical cell-matrix interactions play in regulating biological functions, several microfluidic-based methods have been used to establish 3D collagen gels with defined fiber properties; these gels have helped to establish quantitative relationships between structural ECM cues and observed cell responses. Although existing microfluidic fabrication methods provide excellent definition over collagen fiber anisotropy, they have not demonstrated the independent control over fiber anisotropy and directionality necessary to replicate in vivo collagen architecture. Therefore, to advance collagen microengineering capabilities, we present a user-friendly technology platform that uses controlled fluid flows within a non-uniform microfluidic channel network to create collagen landscapes that can be tuned as a function of extensional strain rate. Herein, we demonstrate capabilities to i) control the degree of fiber anisotropy, ii) create spatial gradients in fiber anisotropy, iii) independently define fiber directionality, and iv) generate multi-material interfaces within a 3D environment. We then address the practical issue of integrating cells into microfluidic systems by using a peel-off template technique to provide direct access to microengineered collagen gels, and demonstrate that cells respond to the defined properties of the landscape. Finally, the platform's modular capability is highlighted by integrating a sub-micrometer thick porous parylene membrane onto the microengineered collagen as a method to define cell-substrate interactions.

Removal of Airway Foreign Body in a Premature Neonate.

OBJECTIVE: Airway foreign bodies are the leading cause of infantile deaths and fourth among preschool children. Airway foreign bodies in extremely premature neonates represent a rare but potentially lethal entity. There are very few reports in the literature describing the treatment of such a condition in premature neonates. The objectives of this report are to describe the safe removal of an airway foreign body in an extremely premature infant using urologic instruments in a trans-endotracheal tube fashion and to review the literature for other techniques that have proven safe and effective, thereby adding technical options for future cases. METHODS: We reviewed the case reported and reviewed pertinent literature. RESULTS: A 2-week old, ex-24 3/7-week, 820-g pre-mature infant was intubated with a 2.5 endotracheal tube. After intubation, a 2-cm foreign body was discovered in the distal trachea by chest x-ray. The child's respiratory status continuously deteriorated with increasing oxygen and positive pressure requirements. While remaining intubated, the child underwent direct suspension laryngoscopy, the ventilator circuitry was disconnected, and the object was successfully removed using a 1.2 mm rigid ureteroscope and 1.1 mm grasping forceps through the 2.5 endotracheal tube. CONCLUSION: This case report demonstrates the effectiveness of using a 1.2 mm ureteroscope and 1.2 mm urologic graspers to extract an airway foreign body from an extremely premature neonate through an endotracheal tube without paralyzing the patient. This method provides a safe and effective means of visualizing and, if necessary, manipulating the airway in a population prone to respiratory complications.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

Screening for Obstructive Sleep Apnea in Children With Sickle Cell Disease: A Pilot Study.

OBJECTIVES/HYPOTHESIS: Obstructive sleep apnea (OSA) and sickle cell disease (SCD) represent two complex disease processes. Current guidelines recommend that children with SCD receive polysomnography (PSG) after presenting with signs or symptoms of sleep-disordered breathing (SDB). Recent studies suggest a disproportionately elevated prevalence of SDB in the population of children with SCD, and traditional risk factors may not be evident within these patients. Further objective testing might be needed to screen all pediatric patients with SCD, even in the absence of overt signs or symptoms of OSA to prevent complications of both conditions. STUDY DESIGN: Prospective cohort study. METHODS: Institutional review board approval was obtained. An eight-question OSA risk assessment screening questionnaire was presented prospectively to 100 consecutive patients with SCD in the pediatric hematology clinic regardless of complaints of SDB. RESULTS: Out of 100 patients, 51 were female. The average age, body mass index (BMI), BMI percentile, and I'M SLEEPY score of the entire cohort were 3.97 years, 15.97%, 55.4%, and 1.63%, respectively. Nineteen patients had a positive sleep apnea screening score and were referred for PSG. The average age BMI, BMI percentile, and I'M SLEEPY score for those 19 patients were 3.77%, 16.67%, 65%, and 3.95%, respectively. Ten patients completed PSG, with seven diagnosed with OSA. CONCLUSIONS: This pilot study demonstrates a higher incidence of SDB and OSA in children with SCD relative to the general pediatric population. Although more PSG reports and further testing is needed to determine whether the results hold, preliminary data indicate that children with SCD should at least undergo OSA screening in the office regardless of overt symptoms. LEVEL OF EVIDENCE: 3 Laryngoscope, 131:E1022-E1028, 2021.

A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.

Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library's usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research.IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library's usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

Effect of drug induced sleep endoscopy on intraoperative decision making in pediatric sleep surgery.

OBJECTIVES: To demonstrate the effect of drug induced sleep endoscopy (DISE) on intra-operative decision making during pediatric sleep surgery for obstructive sleep apnea (OSA). METHODS: A retrospective chart review was performed on pediatric (3-17 years) patients with moderate-to-severe OSA (7.2-71.8) who underwent drug induced sleep endoscopy at the time of initial sleep surgery. The characteristics evaluated included age, race, gender, site of obstruction, type of surgical intervention, pre- and post-operative apnea and hypopnea index. Of the 26 patients that were identified, 18 had both a pre- and post-operative polysomnograms result. RESULTS: All patients underwent DISE immediately prior to surgical treatment. The mean pre-operative AHI for the 18 patients with post-operative polysomnogram results was 21.3 (7.2-71.8). The mean post-operative AHI for the 18 patients was 7.6 (0.7-25.1). There was a significant difference between pre- and post-operative AHI (p < 0.001). Of the 26 patients, the most common area of collapse was the soft palate, occurring in 17/26 (65.4%) patients. Base of tongue involvement was found to be present in 11/26 (42.3%) patients, and the epiglottis was involved in 4/26 (15.4%). Evidence of multilevel collapse was observed in 6/26 (23.1%) patients. Patients observed to have palatal collapse underwent a pharyngoplasty (20/26; 76.9%) at the time of adenotonsillectomy. Three (11.5%) patients underwent a tongue reduction. CONCLUSION: This study provides additional evidence that DISE can affect intra-operative decision making, with the potential for improved post-operative outcomes. A randomized controlled study is needed to determine if these outcomes are better than what can be achieved without DISE.

Quality of life outcomes following velopharyngeal insufficiency surgery.

OBJECTIVES: Velopharyngeal insufficiency (VPI) may be due to functional or anatomic causes, and can lead to speech deficits, communication difficulty, and emotional strain on patients and their caregivers. The VPI Effects on Life Outcomes (VELO) instrument quantifies quality of life outcomes in VPI patients both before and after VPI surgery. This study aims to identify pre-operative patient characteristics associated with better post-operative quality of life. METHODS: This study is a retrospective chart review of 51 patients who underwent VPI surgery between 2009 and 2018 at a tertiary free-standing children's hospital. A 26-item parent-proxy VELO questionnaire was administered by telephone to parents to assess their child's quality of life post-VPI surgery. RESULTS: Twenty-seven parents responded to the VELO questionnaire. Average post-operative VELO score was significantly higher in non-syndromic patients as compared with syndromic patients. Average post-operative VELO score was not significantly different between patients with and without submucous cleft (SMC) or those with mild to moderate versus severe pre-operative hypernasality. On multivariate analysis, absence of genetic syndrome, lack of submucous cleft, and presence of severe-pre-operative hypernasality were significantly and positively associated with increased post-operative VELO scores. CONCLUSION: Children who undergo VPI surgery are more likely to have better post-operative quality of life outcomes if their VPI was not associated with a genetic syndrome or submucous cleft. Non-syndromic and non-SMC patients with severe pre-operative hypernasality may benefit significantly from VPI surgery and have improved post-operative quality of life.

Open Source Brain: A Collaborative Resource for Visualizing, Analyzing, Simulating, and Developing Standardized Models of Neurons and Circuits.

Computational models are powerful tools for exploring the properties of complex biological systems. In neuroscience, data-driven models of neural circuits that span multiple scales are increasingly being used to understand brain function in health and disease. But their adoption and reuse has been limited by the specialist knowledge required to evaluate and use them. To address this, we have developed Open Source Brain, a platform for sharing, viewing, analyzing, and simulating standardized models from different brain regions and species. Model structure and parameters can be automatically visualized and their dynamical properties explored through browser-based simulations. Infrastructure and tools for collaborative interaction, development, and testing are also provided. We demonstrate how existing components can be reused by constructing new models of inhibition-stabilized cortical networks that match recent experimental results. These features of Open Source Brain improve the accessibility, transparency, and reproducibility of models and facilitate their reuse by the wider community.

Connectome to behaviour: modelling Caenorhabditis elegans at cellular resolution.

It has been 30 years since the 'mind of the worm' was published in Philosophical Transactions B (White et al 1986 Phil. Trans. R. Soc. Lond. B314, 1-340). Predicting Caenorhabditis elegans' behaviour from its wiring diagram has been an enduring challenge since then. This special theme issue of Philosophical Transactions B combines research from neuroscientists, physicists, mathematicians and engineers to discuss advances in neural activity imaging, behaviour quantification and multiscale simulations, and how they are bringing the goal of whole-animal modelling at cellular resolution within reach.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

Three-dimensional simulation of the Caenorhabditis elegans body and muscle cells in liquid and gel environments for behavioural analysis.

To better understand how a nervous system controls the movements of an organism, we have created a three-dimensional computational biomechanical model of the Caenorhabditis elegans body based on real anatomical structure. The body model is created with a particle system-based simulation engine known as Sibernetic, which implements the smoothed particle-hydrodynamics algorithm. The model includes an elastic body-wall cuticle subject to hydrostatic pressure. This cuticle is then driven by body-wall muscle cells that contract and relax, whose positions and shape are mapped from C. elegans anatomy, and determined from light microscopy and electron micrograph data. We show that by using different muscle activation patterns, this model is capable of producing C. elegans-like behaviours, including crawling and swimming locomotion in environments with different viscosities, while fitting multiple additional known biomechanical properties of the animal. This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

c302: a multiscale framework for modelling the nervous system of Caenorhabditis elegans.

The OpenWorm project has the ambitious goal of producing a highly detailed in silico model of the nematode Caenorhabditis elegans A crucial part of this work will be a model of the nervous system encompassing all known cell types and connections. The appropriate level of biophysical detail required in the neuronal model to reproduce observed high-level behaviours in the worm has yet to be determined. For this reason, we have developed a framework, c302, that allows different instances of neuronal networks to be generated incorporating varying levels of anatomical and physiological detail, which can be investigated and refined independently or linked to other tools developed in the OpenWorm modelling toolchain.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

OpenWorm: overview and recent advances in integrative biological simulation of Caenorhabditis elegans.

The adoption of powerful software tools and computational methods from the software industry by the scientific research community has resulted in a renewed interest in integrative, large-scale biological simulations. These typically involve the development of computational platforms to combine diverse, process-specific models into a coherent whole. The OpenWorm Foundation is an independent research organization working towards an integrative simulation of the nematode Caenorhabditis elegans, with the aim of providing a powerful new tool to understand how the organism's behaviour arises from its fundamental biology. In this perspective, we give an overview of the history and philosophy of OpenWorm, descriptions of the constituent sub-projects and corresponding open-science management practices, and discuss current achievements of the project and future directions.This article is part of a discussion meeting issue 'Connectome to behaviour: modelling C. elegans at cellular resolution'.

A binary main-belt comet.

Asteroids are primitive Solar System bodies that evolve both collisionally and through disruptions arising from rapid rotation. These processes can lead to the formation of binary asteroids and to the release of dust, both directly and, in some cases, through uncovering frozen volatiles. In a subset of the asteroids called main-belt comets, the sublimation of excavated volatiles causes transient comet-like activity. Torques exerted by sublimation measurably influence the spin rates of active comets and might lead to the splitting of bilobate comet nuclei. The kilometre-sized main-belt asteroid 288P (300163) showed activity for several months around its perihelion 2011 (ref. 11), suspected to be sustained by the sublimation of water ice and supported by rapid rotation, while at least one component rotates slowly with a period of 16 hours (ref. 14). The object 288P is part of a young family of at least 11 asteroids that formed from a precursor about 10 kilometres in diameter during a shattering collision 7.5 million years ago. Here we report that 288P is a binary main-belt comet. It is different from the known asteroid binaries in its combination of wide separation, near-equal component size, high eccentricity and comet-like activity. The observations also provide strong support for sublimation as the driver of activity in 288P and show that sublimation torques may play an important part in binary orbit evolution.

Unit testing, model validation, and biological simulation.

The growth of the software industry has gone hand in hand with the development of tools and cultural practices for ensuring the reliability of complex pieces of software. These tools and practices are now acknowledged to be essential to the management of modern software. As computational models and methods have become increasingly common in the biological sciences, it is important to examine how these practices can accelerate biological software development and improve research quality. In this article, we give a focused case study of our experience with the practices of unit testing and test-driven development in OpenWorm, an open-science project aimed at modeling Caenorhabditis elegans. We identify and discuss the challenges of incorporating test-driven development into a heterogeneous, data-driven project, as well as the role of model validation tests, a category of tests unique to software which expresses scientific models.

Interrelationship of Structure and Function in Maxillofacial Fractures.

Surgical fixation of maxillofacial fractures can be associated with a myriad of surgical complications. Specific complications correlate with the type of fracture. The authors present a case of multiple maxillofacial fractures, briefly review various types of fractures, and discuss the operative decision-making process. This case report serves as an important reminder that the operative decision-making process should take into account a patient's entire clinical condition.

OpenWorm: an open-science approach to modeling Caenorhabditis elegans.

OpenWorm is an international collaboration with the aim of understanding how the behavior of Caenorhabditis elegans (C. elegans) emerges from its underlying physiological processes. The project has developed a modular simulation engine to create computational models of the worm. The modularity of the engine makes it possible to easily modify the model, incorporate new experimental data and test hypotheses. The modeling framework incorporates both biophysical neuronal simulations and a novel fluid-dynamics-based soft-tissue simulation for physical environment-body interactions. The project's open-science approach is aimed at overcoming the difficulties of integrative modeling within a traditional academic environment. In this article the rationale is presented for creating the OpenWorm collaboration, the tools and resources developed thus far are outlined and the unique challenges associated with the project are discussed.

NeuroLex.org: an online framework for neuroscience knowledge.

The ability to transmit, organize, and query information digitally has brought with it the challenge of how to best use this power to facilitate scientific inquiry. Today, few information systems are able to provide detailed answers to complex questions about neuroscience that account for multiple spatial scales, and which cross the boundaries of diverse parts of the nervous system such as molecules, cellular parts, cells, circuits, systems and tissues. As a result, investigators still primarily seek answers to their questions in an increasingly densely populated collection of articles in the literature, each of which must be digested individually. If it were easier to search a knowledge base that was structured to answer neuroscience questions, such a system would enable questions to be answered in seconds that would otherwise require hours of literature review. In this article, we describe NeuroLex.org, a wiki-based website and knowledge management system. Its goal is to bring neurobiological knowledge into a framework that allows neuroscientists to review the concepts of neuroscience, with an emphasis on multiscale descriptions of the parts of nervous systems, aggregate their understanding with that of other scientists, link them to data sources and descriptions of important concepts in neuroscience, and expose parts that are still controversial or missing. To date, the site is tracking ~25,000 unique neuroanatomical parts and concepts in neurobiology spanning experimental techniques, behavioral paradigms, anatomical nomenclature, genes, proteins and molecules. Here we show how the structuring of information about these anatomical parts in the nervous system can be reused to answer multiple neuroscience questions, such as displaying all known GABAergic neurons aggregated in NeuroLex or displaying all brain regions that are known within NeuroLex to send axons into the cerebellar cortex.