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.

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.

Telehealth examination of the lumbar spine.

BACKGROUND: Telehealth evaluations of musculoskeletal conditions have increased due to the stay-at-home policies enacted during the COVID-19 pandemic. Back pain is one of the most common complaints in primary care. While telehealth may never supplant in-person evaluation of back pain, it is imperative in a changing world to learn to perform this evaluation via telephone or video. Virtual visits rely on history-taking and patient self-reported descriptions of pain elicited from self-palpation or specific movements while on the telephone with the clinician. Video examinations provide a unique way of evaluating the lower back compared to telephone because of the ability to visualize the actions of the patient. OBJECTIVE: To create an evaluation pathway for examination of the lumbar spine via telehealth. METHODS: Our group has created a step-by-step evaluation pathway to help physicians direct their patients through typical lumbar examination elements, including inspection, palpation, range of motion, and strength, special, and functional testing. RESULTS: We have developed a table of questions and instructions and a glossary of images of each maneuver to facilitate lumbar spine examination via telemedicine. CONCLUSIONS: This paper provides a guide for extracting clinically relevant information while performing telemedicine examinations of the lumbar spine.

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.

There's Danger in the Drops: Systemic Effects of Ophthalmic Drops Used to Treat Glaucoma.

Glaucoma is a common eye disorder and an irreversible cause of blindness worldwide. There are several treatment options for this condition, with the traditional first-line treatment being ophthalmologic drops. Although administered topically, it is associated with inadvertent systemic absorption leading to a potential for both local and systemic side effects. We discuss the case of a 71-year-old male who presented with a complaint of recurring episodes of distressing sensations including lightheadedness, dyspnea, chest pressure, and faintness. His past medical history included congestive heart failure, hypertension, hyperlipidemia, Barrett's esophagus, and glaucoma. Upon a thorough review of the patient's medications, it was discovered that he had recently been started on timolol ophthalmic drops. The patient then noted that his symptoms had begun after he started using the eye drops. After we recommended that the patient hold the use of the eye drops, these episodes stopped. When prescribing topical ophthalmologic drops, providers must educate patients on common systemic side effects of such drugs.

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.

Professionalism and inter-communication skills (ICS): a multi-site validity study assessing proficiency in core competencies and milestones in medical learners.

BACKGROUND: Interpersonal and Communication Skills (ICS) and Professionalism milestones are challenging to evaluate during medical training. Paucity in proficiency, direction and validity evidence of assessment tools of these milestones warrants further research. We validated the reliability of the previously-piloted Instrument for Communication skills and Professionalism Assessment (InCoPrA) in medical learners. METHODS: This validity approach was guided by the rigorous Kane's Framework. Faculty-raters and standardized patients (SPs) used their respective InCoPrA sub-component to assess distinctive domains pertinent to ICS and Professionalism through multiple expert-built simulated-scenarios comparable to usual care. Evaluations included; inter-rater reliability of the faculty total score; the correlation between the total score by the SPs; and the average of the total score by two-faculty members. Participants were surveyed regarding acceptability, realism, and applicability of this experience. RESULTS: Eighty trainees and 25 faculty-raters from five medical residency training sites participated. ICC of the total score between faculty-raters was generally moderate (ICC range 0.44-0.58). There was on average a moderate linear relationship between the SPs and faculty total scores (Pearson correlations range 0.23-0.44). Majority of participants ascertained receiving a meaningful, immediate, and comprehensive patient-faculty feedback. CONCLUSIONS: This work substantiated that InCoPrA was a reliable, standardized, evidence-based, and user-friendly assessment tool for ICS and Professionalism milestones. Validating InCoPrA showed generally-moderate agreeability and high acceptability. Using InCoPrA also promoted engaging all stakeholders in medical education and training-faculty, learners, and SPs-using simulation-media as pathway for comprehensive feedback of milestones growth.

Primary Care Perceptions and Practices on Discussion and Advice Regarding Sexual Practices.

OBJECTIVES: The United States has experienced an increase in sexually transmitted infections (STIs) in the past decade, a trend that may be influenced by communication gaps between family physicians and patients. We sought to identify factors that hinder discussion about safe sexual practices and STIs, understand physicians' perceptions of their role in preventing STIs, and explore methods of initiating discussions on sexual health. METHODS: From April 30, 2016 to September 1, 2016, family physicians at our institution were given written surveys with 22 questions to answer and rank in order of their best practice. The survey assessed participants' age, sex, level of medical education, and possible barriers to initiating discussion and offering advice on safe sexual practices. RESULTS: All of the participants identified time constraints and the presence of a patient's spouse, parents, or siblings as the most common barriers. Other barriers included fear of embarrassing patients and feeling inadequately knowledgeable about the sexual practices of lesbian, bisexual, gay, and transgender patients. All of the participants reported that patients rarely object to discussing sexual behaviors. CONCLUSIONS: Our study identified several barriers that family physicians may face when initiating discussions and advising patients on safe sexual practices. To prevent new cases of STIs, it is important to work around these barriers to improve physician-patient communication. This can be further improved by providing continuous learning opportunities for medical students, residents, and board-certified family physicians on ways to appropriately counsel patients on safe sexual practices.

Patient-Centered Medical Home With Colocation: Observations and Insights From an Academic Family Medicine Clinic.

There is a movement in the United States to transform family medicine practices from single physician-based patient care to team-based care. These teams are usually composed of multiple disciplines, including social workers, pharmacists, registered nurses, physician assistants, nurse practitioners, and physicians. The teams support patients and their families, provide holistic care to patients of all ages, and allow their members to work to the highest level of their training in an integrated fashion. Grouping care team members together within visual and auditory distance of each other is likely to enhance communication and teamwork, resulting in more efficient care for patients. This grouping is termed colocation. The authors describe how the use of colocation can lead to clearer, faster communication between care team members. This practice style has the potential to be expanded into various clinical settings in any given health system and to almost all clinical specialties and practices.

Degeneration of Injured Axons and Dendrites Requires Restraint of a Protective JNK Signaling Pathway by the Transmembrane Protein Raw.

The degeneration of injured axons involves a self-destruction pathway whose components and mechanism are not fully understood. Here, we report a new regulator of axonal resilience. The transmembrane protein Raw is cell autonomously required for the degeneration of injured axons, dendrites, and synapses in Drosophila melanogaster In both male and female raw hypomorphic mutant or knock-down larvae, the degeneration of injured axons, dendrites, and synapses from motoneurons and sensory neurons is strongly inhibited. This protection is insensitive to reduction in the levels of the NAD+ synthesis enzyme Nmnat (nicotinamide mononucleotide adenylyl transferase), but requires the c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase and the transcription factors Fos and Jun (AP-1). Although these factors were previously known to function in axonal injury signaling and regeneration, Raw's function can be genetically separated from other axonal injury responses: Raw does not modulate JNK-dependent axonal injury signaling and regenerative responses, but instead restrains a protective pathway that inhibits the degeneration of axons, dendrites, and synapses. Although protection in raw mutants requires JNK, Fos, and Jun, JNK also promotes axonal degeneration. These findings suggest the existence of multiple independent pathways that share modulation by JNK, Fos, and Jun that influence how axons respond to stress and injury.SIGNIFICANCE STATEMENT Axonal degeneration is a major feature of neuropathies and nerve injuries and occurs via a cell autonomous self-destruction pathway whose mechanism is poorly understood. This study reports the identification of a new regulator of axonal degeneration: the transmembrane protein Raw. Raw regulates a cell autonomous nuclear signaling pathway whose yet unknown downstream effectors protect injured axons, dendrites, and synapses from degenerating. These findings imply that the susceptibility of axons to degeneration is strongly regulated in neurons. Future understanding of the cellular pathway regulated by Raw, which engages the c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase and Fos and Jun transcription factors, may suggest new strategies to increase the resiliency of axons in debilitating neuropathies.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

Urinary Tract Infection Antibiotic Resistance in the United States.

Urinary tract infection (UTI) is one of the most common entities in medicine and affected patients present daily in a typical family medicine practice. The patients often present with the "classic symptoms" of dysuria and increased frequency, but sometimes they are asymptomatic or have a mixed picture. In most cases, an antibiotic is prescribed, and this practice is likely contributing to increasing worldwide antibiotic resistance. To help combat this problem, it is important that clinicians seek out their local bacterial resistance patterns and antibiograms, properly diagnose and treat UTI if indicated, and recognize their role in antibiotic stewardship.

Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene-mediated silencing.

Mod5 is a multifunctional protein that modifies a subset of tRNAs in the cytoplasm and is also required for an RNA-mediated form of transcriptional silencing. Previous in vivo studies have shown that the nuclear silencing function of Mod5 does not require that the causative tRNA gene encode a Mod5 substrate, although Mod5 is still required. However, previous data have not directly tested whether Mod5 can directly bind substrate and nonsubstrate RNAs. We herein demonstrate that Mod5 directly binds to both substrate and nonsubstrate RNAs, including a highly structured, non-tRNA sequence (5S-rRNA), consistent with previous in vivo data. Furthermore, we show that some RNAs drastically change the aggregation behavior of Mod5 with implications for tRNA gene-mediated silencing.

Site-specific, covalent incorporation of Tus, a DNA-binding protein, on ionic-complementary self-assembling peptide hydrogels using transpeptidase Sortase A as a conjugation tool†Dedicated to the memory of Joachim H. G. Steinke.‡Electronic supplementary information (ESI) available: Further experimental data. See DOI: 10.1039/c3sm00131hClick here for additional data file.

The site-specific conjugation of DNA-binding protein (Tus) to self-assembling peptide FEFEFKFKK was demonstrated. Rheology studies and TEM of the corresponding hydrogels (including PNIPAAm-containing systems) showed no significant variation in properties and hydrogel morphology compared to FEFEFKFKK. Critically, we demonstrate that Tus is accessible within the gel network displaying DNA-binding properties.

Fibrin-loaded porous poly(ethylene glycol) hydrogels as scaffold materials for vascularized tissue formation.

Vascular network formation within biomaterial scaffolds is essential for the generation of properly functioning engineered tissues. In this study, a method is described for generating composite hydrogels in which porous poly(ethylene glycol) (PEG) hydrogels serve as scaffolds for mechanical and structural support, and fibrin is loaded within the pores to induce vascularized tissue formation. Porous PEG hydrogels were generated by a salt leaching technique with 100-150-µm pore size and thrombin (Tb) preloaded within the scaffold. Fibrinogen (Fg) was loaded into pores with varying concentrations and polymerized into fibrin due to the presence of Tb, with loading efficiencies ranging from 79.9% to 82.4%. Fibrin was distributed throughout the entire porous hydrogels, lasted for greater than 20 days, and increased hydrogel mechanical stiffness. A rodent subcutaneous implant model was used to evaluate the influence of fibrin loading on in vivo response. At weeks 1, 2, and 3, all hydrogels had significant tissue invasion, but no difference in the depth of invasion was found with the Fg concentration. Hydrogels with fibrin loading induced more vascularization, with a significantly higher vascular density at 20 mg/mL (week 1) and 40 mg/mL (weeks 2 and 3) Fg concentration compared to hydrogels without fibrin. In conclusion, we have developed a composite hydrogel that supports rapid vascularized tissue ingrowth, and thus holds great potential for tissue engineering applications.

Henoch-Schönlein purpura: a review article.

Henoch-Schönlein purpura (HSP) is the most common vasculitis of childhood. Although HSP is typically a disease of children, adult cases have been described. HSP can affect multiple organs with a characteristic rash present in all patients. Most cases resolve with symptomatic treatment, but serious complications can occur such as renal failure. Primary care physicians should be well aware of the disease because the true incidence is probably underestimated.