Chloride-dependent mechanisms of multimodal sensory discrimination and nociceptive sensitization in Drosophila.

Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval class III (CIII) neurons are peripheral noxious cold nociceptors and innocuous touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69-a fly homologue of NKCC1-results in phenotypes consistent with neuropathic sensitization, including behavioral sensitization and neuronal hyperexcitability, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

Columbia Icefield Bus Rollover: A Case Study of Wilderness Mass Casualty Triage, Treatment, and Transport.

Mass casualty incidents (MCIs) are rare in wilderness and mountain settings. Few case studies have reported the response of such events within jurisdictions with well-developed trauma and emergency medical services systems (EMS). Here we explore a MCI in a wilderness setting on the Columbia Icefield inside the Jasper National Park within the Canadian Rocky Mountains. An all-terrain bus was involved that had rolled over while transporting tourists to explore the glacier. The bus rolled multiple times down the slope adjacent to the road, leading to 3 deceased and 21 patients requiring transport. A massive pre-hospital response ensued.Due to the location, extreme environment, and unusual complexities, the response involved significant use of aeromedical resources, physician field deployment, and centralized coordination centers. Readers are reminded of the importance of aeromedical surge capacity in allowing for effective distribution of patients to multiple receiving facilities. Our experience aligns with and reinforces many of the recommendations for wilderness MCI management; however, future research should focus on determining optimal triage strategies for mountain MCIs. Furthermore, future research should explore optimal strategies for developing a rescue chain given the availability of mixed transport resources, as well as the role of physicians in MCI response and where they are best placed in the incident command system.

Closed Reduction of Anterior Shoulder Dislocations Performed by Ski Patrollers in the Alpine Prehospital Environment: A Retrospective Review Demonstrating Efficacy in a Canadian Ski Resort.

INTRODUCTION: Shoulder dislocations are common ski hill injuries. Rapid reduction is known to improve outcomes; however, advanced providers are not always available to provide care to these patients. In 2017, nonmedical ski patrollers at Sunshine Village ski resort in Alberta, Canada, were trained to perform anterior shoulder dislocation (ASD) reductions. Program success was determined by a chart review after the 2020 ski season. METHODS: This study retrospectively reviewed data on patients who presented to Sunshine Village ski patrol with a suspected ASD and who met the study inclusion criteria from November 2017 through March 2020. Data were collected from ski patrol electronic patient care records regarding general demographics, reduction technique used, analgesia administration, and reduction success rates. RESULTS: Ninety-six cases were available for review after exclusions. Trained nonmedical ski patrollers successfully reduced 82 of these cases, resulting in an overall reduction success rate of 89%. Sixty-three (66%) of these patients had experienced first-time dislocations. Eighty-two (87%) patients were male, with a median age of 25 y. The most used technique was the Cunningham method (75%), and analgesia was administered to 70% of patients. CONCLUSIONS: This retrospective study documents the results of a quality assurance review of the treatment of ASD at Sunshine Village ski resort. With a success rate of 89%, the evidence supports the conclusion that nonmedical ski patrollers can successfully perform ASD reductions. We believe training ski patrollers to reduce ASD improved patient care in our austere environment by providing early definitive treatment with a high success rate.

Formin 3 directs dendritic architecture via microtubule regulation and is required for somatosensory nociceptive behavior.

Dendrite shape impacts functional connectivity and is mediated by organization and dynamics of cytoskeletal fibers. Identifying the molecular factors that regulate dendritic cytoskeletal architecture is therefore important in understanding the mechanistic links between cytoskeletal organization and neuronal function. We identified Formin 3 (Form3) as an essential regulator of cytoskeletal architecture in nociceptive sensory neurons in Drosophila larvae. Time course analyses reveal that Form3 is cell-autonomously required to promote dendritic arbor complexity. We show that form3 is required for the maintenance of a population of stable dendritic microtubules (MTs), and mutants exhibit defects in the localization of dendritic mitochondria, satellite Golgi, and the TRPA channel Painless. Form3 directly interacts with MTs via FH1-FH2 domains. Mutations in human inverted formin 2 (INF2; ortholog of form3) have been causally linked to Charcot-Marie-Tooth (CMT) disease. CMT sensory neuropathies lead to impaired peripheral sensitivity. Defects in form3 function in nociceptive neurons result in severe impairment of noxious heat-evoked behaviors. Expression of the INF2 FH1-FH2 domains partially recovers form3 defects in MTs and nocifensive behavior, suggesting conserved functions, thereby providing putative mechanistic insights into potential etiologies of CMT sensory neuropathies.

Identification of a neural basis for cold acclimation in Drosophila larvae.

Low temperatures can be fatal to insects, but many species have evolved the ability to cold acclimate, thereby increasing their cold tolerance. It has been previously shown that Drosophila melanogaster larvae perform cold-evoked behaviors under the control of noxious cold-sensing neurons (nociceptors), but it is unknown how the nervous system might participate in cold tolerance. Herein, we describe cold-nociceptive behavior among 11 drosophilid species; we find that the predominant cold-evoked larval response is a head-to-tail contraction behavior, which is likely inherited from a common ancestor, but is unlikely to be protective. We therefore tested the hypothesis that cold nociception functions to protect larvae by triggering cold acclimation. We found that Drosophila melanogaster Class III nociceptors are sensitized by and critical to cold acclimation and that cold acclimation can be optogenetically evoked, sans cold. Collectively, these findings demonstrate that cold nociception constitutes a peripheral neural basis for Drosophila larval cold acclimation.

Dissecting the Molecular and Neural Circuit Bases of Behavior as an Introduction to Discovery-Driven Research; A Report on a Course-Based Undergraduate Research Experience.

Herein we discuss a Course-Based Undergraduate Research Experience (CURE) developed in order to engage novice undergraduates in active learning and discovery-driven original research. This course leverages the powerful genetic toolkits available for Drosophila melanogaster in order to investigate the cellular and molecular bases of cold nociception. Given the relatively inexpensive nature of Drosophila rearing, a growing suite of publicly available neurogenomic data, large collections of transgenic stocks available through community stock centers, and Drosophila's highly stereotyped behaviors, this CURE design constitutes a cost-effective approach to introduce students to principles and techniques in genetics, genomics, behavioral neuroscience, research design, and scientific presentation. Moreover, we discuss how this paradigm might be adapted for continued use in investigating any number of systems and/or behaviors - a property we posit is key to impactful CURE design.

Drosophila menthol sensitivity and the Precambrian origins of transient receptor potential-dependent chemosensation.

Transient receptor potential (TRP) cation channels are highly conserved, polymodal sensors which respond to a wide variety of stimuli. Perhaps most notably, TRP channels serve critical functions in nociception and pain. A growing body of evidence suggests that transient receptor potential melastatin (TRPM) and transient receptor potential ankyrin (TRPA) thermal and electrophile sensitivities predate the protostome-deuterostome split (greater than 550 Ma). However, TRPM and TRPA channels are also thought to detect modified terpenes (e.g. menthol). Although terpenoids like menthol are thought to be aversive and/or harmful to insects, mechanistic sensitivity studies have been largely restricted to chordates. Furthermore, it is unknown if TRP-menthol sensing is as ancient as thermal and/or electrophile sensitivity. Combining genetic, optical, electrophysiological, behavioural and phylogenetic approaches, we tested the hypothesis that insect TRP channels play a conserved role in menthol sensing. We found that topical application of menthol to Drosophila melanogaster larvae elicits a Trpm- and TrpA1-dependent nocifensive rolling behaviour, which requires activation of Class IV nociceptor neurons. Further, in characterizing the evolution of TRP channels, we put forth the hypotheses that three previously undescribed TRPM channel clades (basal, αTRPM and ßTRPM), as well as TRPs with residues critical for menthol sensing, were present in ancestral bilaterians. This article is part of the Theo Murphy meeting issue 'Evolution of mechanisms and behaviour important for pain'.

Dendritic Cytoskeletal Architecture Is Modulated by Combinatorial Transcriptional Regulation in Drosophila melanogaster.

Transcription factors (TFs) have emerged as essential cell autonomous mediators of subtype specific dendritogenesis; however, the downstream effectors of these TFs remain largely unknown, as are the cellular events that TFs control to direct morphological change. As dendritic morphology is largely dictated by the organization of the actin and microtubule (MT) cytoskeletons, elucidating TF-mediated cytoskeletal regulatory programs is key to understanding molecular control of diverse dendritic morphologies. Previous studies in Drosophila melanogaster have demonstrated that the conserved TFs Cut and Knot exert combinatorial control over aspects of dendritic cytoskeleton development, promoting actin and MT-based arbor morphology, respectively. To investigate transcriptional targets of Cut and/or Knot regulation, we conducted systematic neurogenomic studies, coupled with in vivo genetic screens utilizing multi-fluor cytoskeletal and membrane marker reporters. These analyses identified a host of putative Cut and/or Knot effector molecules, and a subset of these putative TF targets converge on modulating dendritic cytoskeletal architecture, which are grouped into three major phenotypic categories, based upon neuromorphometric analyses: complexity enhancer, complexity shifter, and complexity suppressor. Complexity enhancer genes normally function to promote higher order dendritic growth and branching with variable effects on MT stabilization and F-actin organization, whereas complexity shifter and complexity suppressor genes normally function in regulating proximal-distal branching distribution or in restricting higher order branching complexity, respectively, with spatially restricted impacts on the dendritic cytoskeleton. Collectively, we implicate novel genes and cellular programs by which TFs distinctly and combinatorially govern dendritogenesis via cytoskeletal modulation.

Nonsegmented Negative-Sense RNA Viruses-Structural Data Bring New Insights Into Nucleocapsid Assembly.

Viruses with a nonsegmented negative-sense RNA genome (NNVs) include important human pathogens as well as life-threatening zoonotic viruses. These viruses share a common RNA replication complex, including the genomic RNA and three proteins, the nucleoprotein (N), the phosphoprotein (P), and the RNA-dependent RNA polymerase (L). During genome replication, the RNA polymerase complex first synthesizes positive-sense antigenomes, which in turn serve as template for the production of negative-sense progeny genomes. These newly synthesized antigenomic and genomic RNAs must be encapsidated by N, and the source of soluble, RNA-free N, competent for the encapsidation is a complex between N and P, named the N0-P complex. In this review, we summarize recent progress made in the structural characterization of the different components of this peculiar RNA polymerase machinery. We discuss common features and replication strategies and highlight idiosyncrasies encountered in different viruses, along with the key role of the dual ordered/disordered architecture of protein components and the dynamics of the viral polymerase machinery. In particular, we focus on the N0-P complex and its role in the nucleocapsid assembly process. These new results provide evidence that the mechanism of NC assembly is conserved between the different families and thus support a divergent evolution from a common ancestor. In addition, the successful inhibition of infection due to different NNVs by peptides derived from P suggests that the mechanism of NC assembly is a potential target for antiviral development.

Molecular identification of iridoviruses infecting various sturgeon species in Europe.

Iridoviridae are known to cause disease in sturgeons in North America. Here, histological and molecular methods were used to screen for this family of virus in sturgeons from various European farms with low-to-high morbidity. Some histological samples revealed basophilic cells in the gill and labial epithelia, strongly suggesting the accumulation of iridovirus particles. Newly developed generic PCR tests targeting the major capsid protein (MCP) gene of sturgeon iridoviruses identified in North America, namely the white sturgeon iridovirus and the Namao virus (NV), produced positive signals in most samples from four sturgeon species: Russian (Acipenser gueldenstaedtii), Siberian (A. baerii), Adriatic (A. naccarii) and beluga (Huso huso). The sequences of the PCR products were generally highly similar one another, with nucleotide identities greater than 98%. They were also related to (74-88%), although distinct from, American sturgeon iridoviruses. These European viruses were thus considered variants of a single new virus, provisionally named Acipenser iridovirus-European (AcIV-E). Moreover, three samples infected with AcIV-E showed genetic heterogeneity, with the co-existence of two sequences differing by five nucleotides. One of our European samples carried a virus distinct from AcIV-E, but closely related to NV identified in Canada (95%). This study demonstrates the presence of two distinct sturgeon iridoviruses in Europe: a new genotype AcIV-E and an NV-related virus.

First detection of Cyprinid Herpesvirus 2 (CyHV-2) in goldfish (Carassius auratus) in France.

Massive mortalities of Carassius auratus (L.) occurred in a farm in France during summer 2014. Fish presented anorexia, loss of scales and large amounts of mucus on the gills. Necrosis of the distal tip of the filament and the lamellae, combined with fusion of the lamellae, was observed, as well as necrosis in the hematopoietic organs and in the digestive tract. The histological examination led to hypothesize the implication of a virus in the mortality. The presence of cyprinid herpesvirus 2 (CyHV-2) in dead fish was demonstrated by amplification and sequencing of portions of the DNA polymerase and helicase genes, both sequences exhibiting 100% identity with CyHV-2 from Japan. In an attempt to find genetic markers of variation, two regions containing tandem repeats in the Japanese genome were amplified from a virus-positive sample from the present outbreak. A first region (mB) was fully identical to the Japanese isolate. However, the second region (mA) exhibited a range of deletions and substitutions compared to CyHV-2 from Japan. This is the first report of CyHV-2 in France in association with mortality of goldfish and the first identification of a molecular marker for its tracing.

Ultrasound assessment of gastric volume in the fasted pediatric patient undergoing upper gastrointestinal endoscopy: development of a predictive model using endoscopically suctioned volumes.

BACKGROUND: Aspiration of gastric contents can be a serious anesthetic-related complication. Gastric antral sonography prior to anesthesia may have a role in identifying pediatric patients at risk of aspiration. We examined the relationship between sonographic antral area and endoscopically suctioned gastric volumes, and whether a 3-point qualitative grading system is applicable in pediatric patients. METHODS: Fasted patients presenting to a pediatric hospital for upper gastrointestinal endoscopy were included in the study. Sonographic measurement of the antral cross-sectional area (CSA) in supine (supine CSA) and right lateral decubitus (RLD CSA) position was completed, and the antrum was designated as empty or nonempty. Gastric contents were endoscopically suctioned and measured. Multiple regression analysis was used to fit a mathematical model to estimate gastric volume. RESULTS: One hundred patients (aged 11-216 months) were included. The gastric antrum was measured in 94% and 99% of patients in the supine and RLD positions, respectively. Gastric antral CSA correlated with total gastric volume in both supine (ρ = 0.63) and RLD (ρ = 0.67) positions. A mathematical model incorporating RLD CSA and age (R(2)  = 0.60) was determined as the best-fit model to predict gastric volumes. Increasing gastric antral grade (0-2) was associated with increasing gastric fluid volume. CONCLUSION: The results suggest that sonographic assessment of the gastric antrum provides useful information regarding gastric content (empty versus nonempty) and volume (ml·kg(-1) ) in pediatric patients. Results suggest that the three-point grading system may be a valuable tool to assess gastric 'fullness' based on a qualitative exam of the antrum.

Adult neural stem cells: isolation and propagation.

Individualized therapy using adult stem cells constitutes a revolutionary vision for molecular medicine of the future. The field of stem cell biology has accelerated dramatically such that it now appears feasible to treat an individual patient's disease with native or modified stem cells collected from the same patient. Neurodegenerative disease is a high-priority goal for stem cell therapy due to the tremendous clinical urgency to reduce the worldwide suffering associated with this class of diseases. This chapter focuses on adult neural stem cells as a prototype for the general field of adult stem cell therapy. Studies of the origin and function of neural stem cells reveals that the adult brain can generate new neurons. This finding provides the rationale for the therapeutic application of adult neural stem cells to treat neuronal damage or loss. Experimental progress in treating Parkinson's disease is discussed in some detail as an example of one of the most promising areas for adult neural stem cell therapy. Methods for neural stem cell isolation and propagation are included.

Role of DegP for two-partner secretion in Bordetella.

Sorting of proteins destined to the surface or the extracellular milieu is mediated by specific machineries, which guide the protein substrates towards the proper route of secretion and determine the compartment in which folding occurs. In gram-negative bacteria, the two-partner secretion (TPS) pathway is dedicated to the secretion of large proteins rich in beta-helical structure. The secretion of the filamentous haemagglutinin (FHA), a 230 kDa adhesin of Bordetella pertussis, represents a model TPS system. FHA is exported by the Sec machinery and transits through the periplasm in an extended conformation. From there it is translocated across the outer membrane by its dedicated transporter FhaC to finally fold into a long beta-helix at the cell surface in a progressive manner. In this work, we show that B. pertussis lacking the periplasmic chaperone/protease DegP has a strong growth defect at 37 degrees C, and the integrity of its outer membrane is compromised. While both phenotypes are significantly aggravated by the presence of FHA, the chaperone activity of DegP markedly alleviates the periplasmic stress. In vitro, DegP binds to non-native FHA with high affinity. We propose that DegP chaperones the extended FHA polypeptide in the periplasm and is thus involved in the TPS pathway.

GABA is not elevated during neuroprotective neuronal depression in the hypoxic epaulette shark (Hemiscyllium ocellatum).

Prolonged hypoxic exposure results in cell failure, glutamate excitotoxicity and apoptosis in the brain. The epaulette shark can withstand prolonged hypoxic exposure without brain injury, while maintaining normal function and activity at tropical temperatures. We examined whether the inhibitory neurotransmitter GABA was involved in hypoxia tolerance and neuroprotection during hypoxic preconditioning. Sharks were exposed to either cyclic hypoxic preconditioning or normoxic conditions. Whole brain GABA concentration was determined using high performance liquid chromatography; GABA distribution in neuronal structures was localised with immunohistochemistry and quantified. While the overall brain level of GABA was not significantly different, there was a significant heterogeneous change in GABA distribution. GABA immunoreactivity was elevated in key motor and sensory nuclei from preconditioned animals, including the nucleus motorius nervi vagi and the cerebellar crest (p<0.001), corresponding to areas of previously reported neuronal hypometabolism. Since the neuroprotection in all other hypoxia and anoxia tolerant species examined so far relies in part on significant elevations in GABA and the phylogenetically older epaulette shark does not, it is reasonable to assume that further research in this unique animal model may yield clues to new key modulators of neuroprotection. Understanding such mechanisms may facilitate the development of therapeutic interventions in the treatment of transient ischaemic attacks, strokes and traumatic brain injury.

Direct cerebral oxygenation monitoring--a systematic review of recent publications.

This review has been compiled to assess publications related to the clinical application of direct cerebral tissue oxygenation (PtiO2) monitoring published in international, peer-reviewed scientific journals. Its goal was to extract relevant, i.e. positive and negative information on indications, clinical application, safety issues and impact on clinical situations as well as treatment strategies in neurosurgery, neurosurgical anaesthesiology, neurosurgical intensive care, neurology and related specialties. For completeness' sake it also presents some related basic science research. PtiO2 monitoring technology is a safe and valuable cerebral monitoring device in neurocritical care. Although a randomized outcome study is not available its clinical utility has repeatedly been clearly confirmed because it adds a monitoring parameter, independent from established cerebral monitoring devices. It offers new insights into cerebral physiology and pathophysiology. Pathologic values have been established in peer-reviewed research, which are not only relevant to outcome but are treatable. The benefits clearly outweigh the risks, which remains unchallenged in all publications retrieved. It is particularly attractive because it offers continuous, real-time data and is available at the bedside.

Multimodality monitoring in severe traumatic brain injury: the role of brain tissue oxygenation monitoring.

Traumatic brain injury (TBI) is a major cause of morbidity and mortality with widespread social, personal, and financial implications for those who survive. TBI is caused by four main events: motor vehicle accidents, sporting injuries, falls, and assaults. Similarly to international statistics, annual incidence reports for TBI in Australia are between 100 and 288 per 100,000. Regardless of the cause of TBI, molecular and cellular derangements occur that can lead to neuronal cell death. Axonal transport disruption, ionic disruption, reduced energy formation, glutamate excitotoxicity, and free radical formation all contribute to the complex pathophysiological process of TBI-related neuronal death. Targeted pharmacological therapy has not proved beneficial in improving patient outcome, and monitoring and maintenance of various physiological parameters is the mainstay of current therapy. Parameters monitored include arterial blood pressure, blood gases, intracranial pressure, cerebral perfusion pressure, cerebral blood flow, and direct brain tissue oxygen measurement (ptiO2). Currently, indirect brain oximetry is used for cerebral oxygenation determination, which provides some information regarding global oxygenation levels. A newly developed oximetry technique, has shown promising results for the early detection of cerebral ischemia. ptiO2 monitoring provides a safe, easy, and sensitive method of regional brain oximetry, providing a greater understanding of neurophysiological derangements and the potential for correcting abnormal oxygenation earlier, thus improving patient outcome. This article reviews the current status of bedside monitoring for patients with TBI and considers whether ptiO2 has a role in the modern intensive care setting.

The pKa of His-24 in the folding transition state of apomyoglobin.

In native apomyoglobin, His-24 cannot be protonated, although at pH 4 the native protein forms a molten globule folding intermediate in which the histidine residues are readily protonated. The inability to protonate His-24 in the native protein dramatically affects the unfolding/refolding kinetics, as demonstrated by simulations for a simple model. Kinetic data for wild type and for a mutant lacking His-24 are analyzed. The pK(a) values of histidine residues in native apomyoglobin are known from earlier studies, and the average histidine pK(a) in the molten globule is determined from the pH dependence of the equilibrium between the native and molten globule forms. Analysis of the pH-dependent unfolding/refolding kinetics reveals that the average pK(a) of the histidine residues, including His-24, is closely similar in the folding transition state to the value found in the molten globule intermediate. Consequently, protonation of His-24 is not a barrier to refolding of the molten globule to the native protein. Instead, the normal pK(a) of His-24 in the transition state, coupled with its inaccessibility in the native state, promotes fast unfolding at low pH. The analysis of the wild-type results is confirmed and extended by using the wild-type parameters to fit the unfolding kinetics of a mutant lacking His-24.