Immobilizing Second-Instar Drosophila Larvae for Imaging and Surgery Using the Larva Chip.

The simple body plan and semitranslucent cuticle of the Drosophila larva allow for imaging of structures close to the body wall within intact animals. These include sensory neurons, muscles, neuromuscular junctions, and some regions of the segmental nerve. However, imaging within an intact larva requires a strategy to immobilize the animal in a position that presents the structures within the working distance of the microscope objective. Although various methods have been implemented for Drosophila larvae, this protocol describes a simple and noninvasive method that makes use of the polydimethylsiloxane (PDMS) larva chip. This larva chip immobilizes animals without the use of anesthetics or changes in temperature, which alter neuronal physiology, making it suitable for calcium imaging of endogenous activity in live animals. The membrane is air-permeable. Animals robustly survive short periods of immobilization (up to 30 min) and can even survive longer time periods. Since animals recover well after the procedure, the same animal can be reimaged multiple times. This makes the method amenable to manipulations such as laser microsurgery, photobleaching, and photoconversion followed by imaging of outcomes of these manipulations over time.

Study of Axonal Injury and Degeneration in Drosophila.

A fundamental feature of nervous systems is a highly specified synaptic connectivity between cells and the ability to adaptively change this connectivity through plasticity mechanisms. Plasticity mechanisms are highly relevant for responding to nervous system damage, and studies using nervous system injury paradigms in Drosophila (as well as other model organisms) have revealed conserved molecular pathways that are triggered by axon damage. Simple assays that introduce injuries to axons in either adult flies or larvae have proven to be particularly powerful for uncovering mechanisms of axonal degeneration and clearance. They have also been used to reveal requirements for regrowth of axons and dendrites, as well as signaling pathways that regulate cellular responses to nerve injury. Here we review commonly used and simple to carry out techniques that enable experimenters to study responses to axonal damage in either adult flies (following antennal transection) or larvae (following nerve crush to segmental nerves). Because axons and dendrites in the larval peripheral nervous system can be readily visualized through the translucent cuticle, another versatile method to probe injury responses is to focus high-energy laser light to a small and specific location in the animal. We therefore discuss a method for immobilizing intact larvae for imaging through the cuticle to carry out injury by pulse dye laser, which can be used to generate many different kinds of injuries and directed ablations within intact larvae. These techniques, combined with powerful genetic tools in Drosophila, make the fruit fly an excellent model system for studying the effects of injury and the mechanisms of axon degeneration, synapse plasticity, and immune response.

Peripheral Nerve Crush in Drosophila Larvae.

The long length of axons makes them vulnerable to damage; hence, it is logical that nervous systems have evolved adaptive mechanisms for responding to axon damage. Studies in Drosophila melanogaster have identified evolutionarily conserved molecular pathways that enable axonal degeneration and regeneration of damaged axons and/or dendrites. This protocol describes a simple method for inducing nerve crush injury to motoneuron and sensory neuron axons in the peripheral (segmental) nerves in second- or early third-instar larvae. Small forceps are used to pinch the cuticle at a location that overlays the segmental nerves. Although the connective tissue of the nerves remains intact and the larva survives the injury, single motoneuron and sensory neuron axons incur a break in continuity at the damage site and then undergo Wallerian degeneration distal to the break. This degeneration includes the dismantling of neuromuscular junction (NMJ) synapses formed by the axons that incurred damage. With stereotyped anatomy and accessibility to structural and electrophysiological studies, the larval NMJ is a good model to characterize the cellular changes that occur in synapses undergoing degeneration and to identify conditions that can protect axons and synapses from degeneration.

Wallerian Degeneration and Clearance of Olfactory Receptor Neuron Axons following Drosophila Antennal Transection.

Neurons extend their axons and dendrites over long distances and rely on evolutionarily conserved mechanisms to maintain the cellular structure and function of neurites at a distance from their cell body. Neurites that lose connection with their cell body following damage or stressors to their cytoskeleton undergo a programmed self-destruction process akin to apoptosis but using different cellular machinery, termed Wallerian degeneration. While first described for vertebrate axons by Augustus Waller in 1850, key discoveries of the enzymes that regulate Wallerian degeneration were made through forward genetic screens in Drosophila melanogaster Powerful techniques for genetic manipulation and visualization of single neurons combined with simple methods for introducing axotomy (neuron severing) to certain neuron types in Drosophila have enabled the discovery and study of the cellular machinery responsible for Wallerian degeneration, in addition to mechanisms that enable clearance of the resulting debris. This protocol describes how to study the degeneration and clearance of axons from olfactory receptor neurons (ORNs). These peripheral neurons reside in the antennae and project axons to olfactory glomeruli of the anterior brain. Simple and nonlethal removal of antennae from adult flies causes axotomy of ORNs, and the fate of the injured axons can be readily visualized in a whole-mount dissected brain. This assay takes advantage of well-characterized genetic methods to robustly and specifically label subsets of ORNs. This method of neurite labeling and axotomy was the first axon injury paradigm to be developed in flies and is still regularly used due to its simplicity to perform, dissect, image, and analyze.

Laser Microsurgery in Drosophila Larvae Using the MicroPoint Ablation System.

Laser microsurgery is a robust method to ablate specific cells in the nervous system and probe the functional consequences of their loss in the animal. By introducing focal lesions to small locations in the animal, laser microsurgery also enables disruptions of specific connections within neuronal circuits and the study of how the nervous system responds to precise forms of damage (for instance, damage to specific axons or dendrites, which have been found to evoke different kinds of responses in neurons). The MicroPoint laser is a pulsed dye laser that can be mounted onto any standard microscope, hence is an affordable alternative to two-photon lasers for providing high powered focal ablations. This protocol describes how to use a MicroPoint laser ablation system to induce focal injuries in Drosophila larvae. This protocol guides a user who has access to a MicroPoint laser that has already been installed onto an appropriate microscope for high-resolution imaging and configured for laser ablation using Coumarin 440 dye. The protocol covers how to use the laser to carry out surgeries or ablation, how to change the laser dye and calibrate the power settings, and how to make sure the laser is properly focused. While the protocol provides an example of axotomy (axon severing) in the peripheral nervous system of Drosophila larvae, use of the MicroPoint system can be adapted to other focal surgeries in other organisms.

Sarm1 is not necessary for activation of neuron-intrinsic growth programs yet required for the Schwann cell repair response and peripheral nerve regeneration.

Upon peripheral nervous system (PNS) injury, severed axons undergo rapid SARM1-dependent Wallerian degeneration (WD). In mammals, the role of SARM1 in PNS regeneration, however, is unknown. Here we demonstrate that Sarm1 is not required for axotomy induced activation of neuron-intrinsic growth programs and axonal growth into a nerve crush site. However, in the distal nerve, Sarm1 is necessary for the timely induction of the Schwann cell (SC) repair response, nerve inflammation, myelin clearance, and regeneration of sensory and motor axons. In Sarm1-/- mice, regenerated fibers exhibit reduced axon caliber, defective nerve conduction, and recovery of motor function is delayed. The growth hostile environment of Sarm1-/- distal nerve tissue was demonstrated by grafting of Sarm1-/- nerve into WT recipients. SC lineage tracing in injured WT and Sarm1-/- mice revealed morphological differences. In the Sarm1-/- distal nerve, the appearance of p75NTR+, c-Jun+ SCs is significantly delayed. Ex vivo, p75NTR and c-Jun upregulation in Sarm1-/- nerves can be rescued by pharmacological inhibition of ErbB kinase. Together, our studies show that Sarm1 is not necessary for the activation of neuron intrinsic growth programs but in the distal nerve is required for the orchestration of cellular programs that underlie rapid axon extension.

DLK signaling in axotomized neurons triggers complement activation and loss of upstream synapses.

Axotomized spinal motoneurons (MNs) lose presynaptic inputs following peripheral nerve injury; however, the cellular mechanisms that lead to this form of synapse loss are currently unknown. Here, we delineate a critical role for neuronal kinase dual leucine zipper kinase (DLK)/MAP3K12, which becomes activated in axotomized neurons. Studies with conditional knockout mice indicate that DLK signaling activation in injured MNs triggers the induction of phagocytic microglia and synapse loss. Aspects of the DLK-regulated response include expression of C1q first from the axotomized MN and then later in surrounding microglia, which subsequently phagocytose presynaptic components of upstream synapses. Pharmacological ablation of microglia inhibits the loss of cholinergic C boutons from axotomized MNs. Together, the observations implicate a neuronal mechanism, governed by the DLK, in the induction of inflammation and the removal of synapses.

Exploring Microorganisms from Plastic-Polluted Sites: Unveiling Plastic Degradation and PHA Production Potential.

The exposure of microorganisms to conventional plastics is a relatively recent occurrence, affording limited time for evolutionary adaptation. As part of the EU-funded project BioICEP, this study delves into the plastic degradation potential of microorganisms isolated from sites with prolonged plastic pollution, such as plastic-polluted forests, biopolymer-contaminated soil, oil-contaminated soil, municipal landfill, but also a distinctive soil sample with plastic pieces buried three decades ago. Additionally, samples from Arthropoda species were investigated. In total, 150 strains were isolated and screened for the ability to use plastic-related substrates (Impranil dispersions, polyethylene terephthalate, terephthalic acid, and bis(2-hydroxyethyl) terephthalate). Twenty isolates selected based on their ability to grow on various substrates were identified as Streptomyces, Bacillus, Enterococcus, and Pseudomonas spp. Morphological features were recorded, and the 16S rRNA sequence was employed to construct a phylogenetic tree. Subsequent assessments unveiled that 5 out of the 20 strains displayed the capability to produce polyhydroxyalkanoates, utilizing pre-treated post-consumer PET samples. With Priestia sp. DG69 and Neobacillus sp. DG40 emerging as the most successful producers (4.14% and 3.34% of PHA, respectively), these strains are poised for further utilization in upcycling purposes, laying the foundation for the development of sustainable strategies for plastic waste management.

Opposing roles of Fos, Raw, and SARM1 in the regulation of axonal degeneration and synaptic structure.

Introduction: The degeneration of injured axons is driven by conserved molecules, including the sterile armadillo TIR domain-containing protein SARM1, the cJun N-terminal kinase JNK, and regulators of these proteins. These molecules are also implicated in the regulation of synapse development though the mechanistic relationship of their functions in degeneration vs. development is poorly understood. Results and discussion: Here, we uncover disparate functional relationships between SARM1 and the transmembrane protein Raw in the regulation of Wallerian degeneration and synaptic growth in motoneurons of Drosophila melanogaster. Our genetic data suggest that Raw antagonizes the downstream output MAP kinase signaling mediated by Drosophila SARM1 (dSarm). This relationship is revealed by dramatic synaptic overgrowth phenotypes at the larval neuromuscular junction when motoneurons are depleted for Raw or overexpress dSarm. While Raw antagonizes the downstream output of dSarm to regulate synaptic growth, it shows an opposite functional relationship with dSarm for axonal degeneration. Loss of Raw leads to decreased levels of dSarm in axons and delayed axonal degeneration that is rescued by overexpression of dSarm, supporting a model that Raw promotes the activation of dSarm in axons. However, inhibiting Fos also decreases dSarm levels in axons but has the opposite outcome of enabling Wallerian degeneration. The combined genetic data suggest that Raw, dSarm, and Fos influence each other's functions through multiple points of regulation to control the structure of synaptic terminals and the resilience of axons to degeneration.

Sustainable production and pharmaceutical applications of ß-glucan from microbial sources.

ß-glucans are a large class of complex polysaccharides found in abundant sources. Our dietary sources of ß-glucans are cereals that include oats and barley, and non-cereal sources can consist of mushrooms, microalgae, bacteria, and seaweeds. There is substantial clinical interest in ß-glucans; as they can be used for a variety of diseases including cancer and cardiovascular conditions. Suitable sources of ß-glucans for biopharmaceutical applications include bacteria, microalgae, mycelium, and yeast. Environmental factors including culture medium can influence the biomass and ultimately ß-glucan content. Therefore, cultivation conditions for the above organisms can be controlled for sustainable enhanced production of ß-glucans. This review discusses the various sources of ß-glucans and their cultivation conditions that may be optimised to exploit sustainable production. Finally, this article discusses the immune-modulatory potential of ß-glucans from these sources.

Optimising management of complex regional pain syndrome to improve clinical outcomes throughout the therapy care pathway in England: Protocol for a qualitative interview and observational study with patients and clinicians.

INTRODUCTION: Complex Regional Pain Syndrome (CRPS) is a disabling and distressing chronic pain condition characterised by a range of sensory, motor, autonomic and trophic symptoms. Guidelines recommend early referral for therapies that promote movement of the painful limb. However, evidence suggests a lack of defined therapy pathways for CRPS. AIMS: The current study aims to explore CRPS therapy management in centres of excellence in England, and outside of these settings, to understand what facilitates and hinders best practice. The overall aim is to develop a draft stratified package of care to expedite patient access to optimal CRPS therapy across the management pathway. METHODS AND ANALYSIS: Semi-structured interviews will be conducted with therapists working in CRPS centres of excellence and with therapists in other settings. Observations of therapy interventions in CRPS centres of excellence and interviews with patients who have received this care, will also help to identify potential key care package components. Interview data will be analysed using thematic analysis, mapped to the Theoretical Domains Framework (TDF), and Intervention Mapping Adapt (IMA) framework. Observations will be described and documented using the TDF headings. CONCLUSION: A triangulation protocol for qualitative health research will be used to integrate all data. Online stakeholder events will be held using consensus methods to agree a draft package of care for future implementation following further refinement, testing and evaluation. CLINICAL TRIAL REGISTRATION: The trial was registered with ISRCTN registry on 24 February 2022 (ISRCTN16917807).

'Are Routine Post-discharge Diuretics Necessary After Pediatric Cardiac Surgery?'

A prospective, one-armed, safety non-inferiority trial with historical controls was performed at a single-center, quaternary, children's hospital. Inclusion criteria were children aged 3 months-18 years after pediatric cardiac surgery resulting in a two-ventricle repair between 7/2020 and 7/2021. Eligible patients were compared with patients from a 5-year historical period (selected using a database search). The intervention was that "regular risk" patients received no diuretics and pre-specified "high risk" patients received 5 days of twice per day furosemide at discharge. 61 Subjects received the intervention. None were readmitted for pleural effusions, though 1 subject was treated for a symptomatic pleural effusion with outpatient furosemide. The study was halted after an interim analysis demonstrated that 4 subjects were readmitted with pericardial effusion during the study period versus 2 during the historical control (2.9% versus 0.2%, P = 0.003). We found no evidence that limited post-discharge diuretics results in an increase in readmissions for pleural effusions. This conclusion is limited as not enough subjects were enrolled to definitively show that this strategy is not inferior to the historical practice. There was a statistically significant increase in readmissions for pericardial effusions after implementation of this study protocol which can lead to serious complications and requires further study before conclusions can be drawn regarding optimal diuretic regimens.

Multifaceted roles of SARM1 in axon degeneration and signaling.

Axons are considered to be particularly vulnerable components of the nervous system; impairments to a neuron's axon leads to an effective silencing of a neuron's ability to communicate with other cells. Nervous systems have therefore evolved plasticity mechanisms for adapting to axonal damage. These include acute mechanisms that promote the degeneration and clearance of damaged axons and, in some cases, the initiation of new axonal growth and synapse formation to rebuild lost connections. Here we review how these diverse processes are influenced by the therapeutically targetable enzyme SARM1. SARM1 catalyzes the breakdown of NAD+, which, when unmitigated, can lead to rundown of this essential metabolite and axonal degeneration. SARM1's enzymatic activity also triggers the activation of downstream signaling pathways, which manifest numerous functions for SARM1 in development, innate immunity and responses to injury. Here we will consider the multiple intersections between SARM1 and the injury signaling pathways that coordinate cellular adaptations to nervous system damage.

Current issues and recommendations to manage prescription drug benefits for public health programs.

Higher scrutiny is befalling public payors regarding drug costs and patient access to medications. These issues exist in a complex contractual environment where minimal oversight of pharmacy claim adjudication and reimbursement practices can occur. The complexity of prescription benefits, and the lack of defined expectations or accountability in the system contribute to a sense of frustration by the public. Key areas of improvement for this sector of the health care industry include legislative and regulatory shifts requiring ongoing analyses, reporting, and accountability of pharmacy benefit managers (PBMs) in order to improve payment transparency. These improvements will enable plans to eliminate misaligned incentives in the industry and drive value. Changes in public sector programs should be comprehensive in their approach so that the policy will result in a reduction in costs, enhanced patient access, better patient safety, and improved health outcomes.

Mitochondrial fission, integrity and completion of mitophagy require separable functions of Vps13D in Drosophila neurons.

A healthy population of mitochondria, maintained by proper fission, fusion, and degradation, is critical for the long-term survival and function of neurons. Here, our discovery of mitophagy intermediates in fission-impaired Drosophila neurons brings new perspective into the relationship between mitochondrial fission and mitophagy. Neurons lacking either the ataxia disease gene Vps13D or the dynamin related protein Drp1 contain enlarged mitochondria that are engaged with autophagy machinery and also lack matrix components. Reporter assays combined with genetic studies imply that mitophagy both initiates and is completed in Drp1 impaired neurons, but fails to complete in Vps13D impaired neurons, which accumulate compromised mitochondria within stalled mito-phagophores. Our findings imply that in fission-defective neurons, mitophagy becomes induced, and that the lipid channel containing protein Vps13D has separable functions in mitochondrial fission and phagophore elongation.

Abnormal Microarray, Clinical Outcomes, and Surgical Risk Scores in Young Children with Cardiac Disease.

The clinical implications of abnormal chromosomal microarray (CMA) remain unclear for children less than 1 year of age with critical heart disease. Our objective was to determine whether abnormal CMA was related to surgical severity scores or to pre-determined clinical outcomes, including cardiac arrest. Retrospective review of children under 1 year of age admitted to a pediatric cardiac intensive care unit from December, 2014 to September, 2017. Associations between CMA result and cardiac arrest, syndromic abnormalities, and extracardiac anomalies were evaluated. A simple and multivariable logistic regression model was used to analyze associations between STAT mortality category and CMA result. The overall prevalence of abnormal microarray was 48/168 (29%), with peak prevalence in AV septal defects and left-sided obstructive lesions. There was no statistical association between surgical severity scores and abnormal CMA (STAT 1/2 vs. 3+, odds ratio 0.56, p = 0.196). Abnormal CMA was associated with a higher prevalence of cardiac arrest (5/48 abnormal CMA vs. 2/120 normal CMA, p = 0.02). Abnormal CMA was associated with a higher frequency of syndromic abnormalities (18/48 abnormal CMA vs. 13/120 normal CMA, p < 0.001). There was a high prevalence of abnormal CMA findings in the pediatric cardiac population less than 1 year of age (29%), associated with cardiac arrest, but not associated with surgical risk score. The absence of a standardized protocol for ordering a CMA in the setting of congenital heart disease results in a highly variable prevalence data.

An NAD+/NMN balancing act by SARM1 and NMNAT2 controls axonal degeneration.

Axonal degeneration is controlled by the TIR domain NADase SARM1. In this issue of Neuron, Figley et al. (2021) reveal a key regulatory mechanism that controls SARM1's enzymatic activity, providing insight into how NAD+ biosynthesis by the NMNAT2 enzyme protects axons, and a new therapeutic path to tune SARM1 activity.

Regulation of longevity by depolarization-induced activation of PLC-ß-IP3R signaling in neurons.

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cß (PLC-ß) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-ß activity led to greater release of endoplasmic reticulum Ca2+ via the inositol trisphosphate receptor (IP3R), increased mitochondrial Ca2+ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-ß-IP3R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca2+ into endolysosomes was an intermediary in the regulation of lifespan by IP3Rs. Manipulations that either lowered PLC-ß/IP3R abundance or attenuated endolysosomal Ca2+ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-ß-IP3R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca2+ overload.

Non-Lethal Sequential Individual Monitoring of Viremia in Relation to DNA Vaccination in Fish-Example Using a Salmon Alphavirus DNA Vaccine in Atlantic Salmon Salmo salar.

Traditionally, commercial testing for vaccine efficacy has relied on the mass infection of vaccinated and unvaccinated animals and the comparison of mortality prevalence and incidence. For some infection models where disease does not cause mortality this approach to testing vaccine efficacy is not useful. Additionally, in fish experimental studies on vaccine efficacy and immune response the norm is that several individuals are lethally sampled at sequential timepoints, and results are extrapolated to represent the kinetics of immune and disease parameters of an individual fish over the entire experimental infection period. In the present study we developed a new approach to vaccine testing for viremic viruses in fish by following the same individuals over the course of a DNA vaccination and experimental infection through repeated blood collection and analyses. Injectable DNA vaccines are particularly efficient against viral disease in fish. To date, two DNA vaccines have been authorised for use in fish farming, one in Canada against Infectious Haemorrhagic Necrotic virus and more recently one in Europe against Salmon Pancreatic Disease virus (SPDv) subtype 3. In the current study we engineered and used an experimental DNA vaccine against SPDv subtype 1. We measured viremia using a reporter cell line system and demonstrated that the viremia phase was completely extinguished following DNA vaccination. Differences in viremia infection kinetics between fish in the placebo group could be related to subsequent antibody levels in the individual fish, with higher antibody levels at terminal sampling in fish showing earlier viremia peaks. The results indicate that sequential non-lethal sampling can highlight associations between infection traits and immune responses measured at asynchronous timepoints and, can provide biological explanations for variation in data. Similar to results observed for the SPDv subtype 3 DNA vaccine, the SPDv subtype 1 DNA vaccine also induced an interferon type 1 response after vaccination and provided high protection against SPDv under laboratory conditions when fish were challenged at 7 weeks post-vaccination.