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.

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.

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.

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.

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.

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.

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.

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'.

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.

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.

Two forms of the pH 4 folding intermediate of apomyoglobin.

The pH 4 folding intermediate of apomyoglobin exists in two forms (Ia, Ib) at equilibrium. Their ratio depends on pH, urea concentration and the presence or absence of a stabilizing anion (citrate, sulfate), and it does not depend on protein concentration. The Ia and Ib species are separated by a kinetic barrier and their interconversion can be monitored by tryptophan fluorescence in stopped-flow experiments. At pH 4.2, Ib is converted to Ia at low urea concentrations and urea unfolding gives the unfolding transition of Ia. During the refolding of native (N) apomyoglobin at pH 6, starting from the acid unfolded species (U), both Ia and Ib appear as transient intermediates and both Ia and Ib appear as transient intermediates in the acid-induced unfolding of N. The results are consistent with a linear folding and unfolding pathway: U reversible Ia reversible Ib reversible N. Apomyoglobin provides the opportunity to investigate at equilibrium the structures and properties of two different kinetic folding intermediates. A non-obligatory dimeric species of the pH 4 intermediate is formed slowly and contributes to the refolding kinetics at concentrations above 5 microM. The dimer dissociates slowly and during refolding at pH 6 it forms N in a later time range than does the monomer.

A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: application to ribonuclease A.

A modified pulse-chase experiment is applied to determine if the native-like intermediate IN of ribonuclease A is on or off-pathway. The 1H label retained in the native protein is compared when separate samples of 1H-labeled IN and unfolded protein are allowed to fold to native in identical conditions. The solvent is 2H2O and the pH* is such that the unfolded protein rapidly exchanges its peptide NH protons with solvent, and IN does not. If IN is on-pathway, more 1H-label will be retained in the test sample starting with IN than in the control sample starting with unfolded protein. The results show that IN is a productive (on-pathway) intermediate. Application of the modified pulse-chase experiment to the study of rapidly formed folding intermediates may be possible when a rapid mixing device is used.

Submillisecond unfolding kinetics of apomyoglobin and its pH 4 intermediate.

Submillisecond mixing experiments and tryptophan fluorescence spectroscopy are used to address two questions raised in earlier stopped-flow studies of the folding and unfolding kinetics of sperm whale apomyoglobin. A study of the pH 4 folding intermediate (I) revealed, surprisingly, that its folding and unfolding kinetics are measurable and fit the two-state model except for a possible burst phase in unfolding. Submillisecond mixing experiments confirm the unfolding burst phase and show that its properties are consistent with the recently discovered interconversion between two forms of I, Ia equilibrium Ib. In urea-induced unfolding, Ib is converted to Ia before Ia unfolds, and the unfolding kinetics of Ia fit the two-state model when the burst phase is assigned to Ib-->Ia. The second question is whether the Ia, Ib intermediates accumulate transiently when the native protein (N) unfolds to the acid unfolded form (U). Earlier work showed that Ia and Ib accumulate when U refolds to N at pH 6.0 and the results fit the linear folding pathway U equilibrium Ia equilibrium Ib equilibrium N. We report here that either or both Ia and Ib accumulate transiently when N unfolds to U at pH 2.7 and that the position of the rate-limiting step in the pathway changes between unfolding at pH 2. 7 and refolding at pH 6.0. In unfolding as in refolding, we do not detect a fast track that bypasses the Ia, Ib intermediates.

Thermal unfolding of an intermediate is associated with non-Arrhenius kinetics in the folding of hen lysozyme.

A variety of techniques, including quenched-flow hydrogen exchange labelling monitored by electrospray ionization mass spectrometry, and stopped-flow absorbance, fluorescence and circular dichroism spectroscopy, has been used to investigate the refolding kinetics of hen lysozyme over a temperature range from 2 degrees C to 50 degrees C. Simple Arrhenius behaviour is not observed, and although the overall rate of folding increases from 2 to 40 degrees C, it decreases above 40 degrees C. In addition, the transient intermediate on the major folding pathway at 20 degrees C, in which the alpha-domain is persistently structured in the absence of a stable beta-domain, is thermally unfolded in a sigmoidal transition (T(m) approximately 40 degrees C) indicative of a cooperatively folded state. At all temperatures, however, there is evidence for fast ( approximately 25 %) and slow ( approximately 75 %) populations of refolding molecules. By using transition state theory, the kinetic data from various experiments were jointly fitted to a sequential three-state model for the slow folding pathway. Together with previous findings, these results indicate that the alpha-domain intermediate is a productive species on the folding route between the denatured and native states, and which accumulates as a consequence of its intrinsic stability. Our analysis suggests that the temperature dependence of the rate constant for lysozyme folding depends on both the total change in the heat capacity between the ground and transition states (the dominant factor at low temperatures) and the heat-induced destabilization of the alpha-domain intermediate (the dominant factor at high temperatures). Destabilization of such kinetically competent intermediate species is likely to be a determining factor in the non-Arrhenius temperature dependence of the folding rate of those proteins for which one or more intermediates are populated.

The unfolding enthalpy of the pH 4 molten globule of apomyoglobin measured by isothermal titration calorimetry.

The unfolding enthalpy of the pH 4 molten globule from sperm whale apomyoglobin has been measured by isothermal titration calorimetry, using titration to acid pH. The unfolding enthalpy is close to zero at 20 degrees C, in contrast both to the positive values expected for peptide helices and the negative values reported for holomyoglobin and native apomyoglobin. At 20 degrees C, the hydrophobic interaction should make only a small contribution to the unfolding enthalpy according to the liquid hydrocarbon model. Our result indicates that some factor present in the unfolding enthalpies of native proteins makes the unfolding enthalpy of the pH 4 molten globule less positive than expected from data for peptide helices.

Penicillin binding protein 2x as a major contributor to intrinsic beta-lactam resistance of Streptococcus pneumoniae.

The production and purification to protein homogeneity of a soluble form of PBP2x from a cefotaxime-resistant Streptococcus pneumoniae strain is reported. It was obtained by a site-directed deletion of the membrane anchor in the corresponding gene, a method similar to that successfully utilized for the production of PBP2x from a cefotaxime-sensitive wild type strain. The kinetic parameters characterizing the interactions of both cefotaxime-resistant and -sensitive proteins have been determined and compared. The results are in agreement with the identification of PBP2x as the primary target for cefotaxime in the sensitive strain and as probably one of several targets in the resistant strain.