Calcium dynamics at the neural cell primary cilium regulate Hedgehog signaling-dependent neurogenesis in the embryonic neural tube.

The balance between neural stem cell proliferation and neuronal differentiation is paramount for the appropriate development of the nervous system. Sonic hedgehog (Shh) is known to sequentially promote cell proliferation and specification of neuronal phenotypes, but the signaling mechanisms responsible for the developmental switch from mitogenic to neurogenic have remained unclear. Here, we show that Shh enhances Ca2+ activity at the neural cell primary cilium of developing Xenopus laevis embryos through Ca2+ influx via transient receptor potential cation channel subfamily C member 3 (TRPC3) and release from intracellular stores in a developmental stage-dependent manner. This ciliary Ca2+ activity in turn antagonizes canonical, proliferative Shh signaling in neural stem cells by down-regulating Sox2 expression and up-regulating expression of neurogenic genes, enabling neuronal differentiation. These discoveries indicate that the Shh-Ca2+-dependent switch in neural cell ciliary signaling triggers the switch in Shh action from canonical-mitogenic to neurogenic. The molecular mechanisms identified in this neurogenic signaling axis are potential targets for the treatment of brain tumors and neurodevelopmental disorders.

Signal amplification in growth cone gradient sensing by a double negative feedback loop among PTEN, PI(3,4,5)P3 and actomyosin.

Axon guidance during neural wiring involves a series of precisely controlled chemotactic events by the motile axonal tip, the growth cone. A fundamental question is how neuronal growth cones make directional decisions in response to extremely shallow gradients of guidance cues with exquisite sensitivity. Here we report that nerve growth cones possess a signal amplification mechanism during gradient sensing process. In neuronal growth cones of Xenopus spinal neurons, phosphatidylinositol-3,4,5-trisphosphate (PIP3), an important signaling molecule in chemotaxis, was actively recruited to the up-gradient side in response to an external gradient of brain-derived neurotrophic factor (BDNF), resulting in an intracellular gradient with approximate 30-fold amplification of the input. Furthermore, a reverse gradient of phosphatase and tensin homolog (PTEN) was induced by BDNF within the growth cone and the increased PTEN activity at the down-gradient side is required for the amplification of PIP3 signals. Mechanistically, the establishment of both positive PIP3 and reverse PTEN gradients depends on the filamentous actin network. Together with computational modeling, our results revealed a double negative feedback loop among PTEN, PIP3 and actomyosin for signal amplification, which is essential for gradient sensing of neuronal growth cones in response to diffusible cues.

Growth at Cold Temperature Increases the Number of Motor Neurons to Optimize Locomotor Function.

During vertebrate development, spinal neurons differentiate and connect to generate a system that performs sensorimotor functions critical for survival. Spontaneous Ca2+ activity regulates different aspects of spinal neuron differentiation. It is unclear whether environmental factors can modulate this Ca2+ activity in developing spinal neurons to alter their specialization and ultimately adjust sensorimotor behavior to fit the environment. Here, we show that growing Xenopus laevis embryos at cold temperatures results in an increase in the number of spinal motor neurons in larvae. This change in spinal cord development optimizes the escape response to gentle touch of animals raised in and tested at cold temperatures. The cold-sensitive channel TRPM8 increases Ca2+ spike frequency of developing ventral spinal neurons, which in turn regulates expression of the motor neuron master transcription factor HB9. TRPM8 is necessary for the increase in motor neuron number of animals raised in cold temperatures and for their enhanced sensorimotor behavior when tested at cold temperatures. These findings suggest the environment modulates neuronal differentiation to optimize the behavior of the developing organism.

A successful management after preterm delivery in a patient with severe sepsis during third-trimester pregnancy.

A 33-year-old woman visited the emergency department presenting with fever and dyspnea. She was pregnant with gestational age of 31 weeks and 6 days. She had dysuria for 7 days, and fever and dyspnea for 1 day. The vital signs were as follows: blood pressure 110/70 mmHg, heart rate 118 beats/minute, respiratory rate 28/minute, body temperature 38.7℃, and oxygen saturation by pulse oximetry 84% during inhalation of 5 liters of oxygen by nasal prongs. Crackles were heard over both lung fields. There were no signs of uterine contractions. Chest X-ray and chest computed tomography scan showed multiple consolidations and air bronchograms in both lungs. According to urinalysis, there was pyuria and microscopic hematuria. She was diagnosed with community-acquired pneumonia and urinary tract infection (UTI) that progressed to severe sepsis and acute respiratory failure. We found extended-spectrum beta-lactamase producing Escherichia coli in the blood culture and methicillin-resistant Staphylococcus aureus in the sputum culture. The patient was transferred to the intensive care unit with administration of antibiotics and supplementation of high-flow oxygen. On hospital day 2, hypoxemia was aggravated. She underwent endotracheal intubation and mechanical ventilation. After 3 hours, fetal distress was suspected. Under 100% fraction of inspired oxygen, her oxygen partial pressure was 87 mmHg in the arterial blood. She developed acute kidney injury and thrombocytopenia. We diagnosed her with multi-organ failure due to severe sepsis. After an emergent cesarean section, pneumonia, UTI, and other organ failures gradually recovered. The patient and baby were discharged soon thereafter.

Rapid Response Systems Reduce In-Hospital Cardiopulmonary Arrest: A Pilot Study and Motivation for a Nationwide Survey.

BACKGROUND: Early recognition of the signs and symptoms of clinical deterioration could diminish the incidence of cardiopulmonary arrest. The present study investigates outcomes with respect to cardiopulmonary arrest rates in institutions with and without rapid response systems (RRSs) and the current level of cardiopulmonary arrest rate in tertiary hospitals. METHODS: This was a retrospective study based on data from 14 tertiary hospitals. Cardiopulmonary resuscitation (CPR) rate reports were obtained from each hospital to include the number of cardiopulmonary arrest events in adult patients in the general ward, the annual adult admission statistics, and the structure of the RRS if present. RESULTS: Hospitals with RRSs showed a statistically significant reduction of the CPR rate between 2013 and 2015 (odds ratio [OR], 0.731; 95% confidence interval [CI], 0.577 to 0.927; P = 0.009). Nevertheless, CPR rates of 2013 and 2015 did not change in hospitals without RRS (OR, 0.988; 95% CI, 0.868 to 1.124; P = 0.854). National university-affiliated hospitals showed less cardiopulmonary arrest rate than private university-affiliated in 2015 (1.92 vs. 2.40; OR, 0.800; 95% CI, 0.702 to 0.912; P = 0.001). High-volume hospitals showed lower cardiopulmonary arrest rates compared with medium-volume hospitals in 2013 (1.76 vs. 2.63; OR, 0.667; 95% CI, 0.577 to 0.772; P < 0.001) and in 2015 (1.55 vs. 3.20; OR, 0.485; 95% CI, 0.428 to 0.550; P < 0.001). CONCLUSIONS: RRSs may be a feasible option to reduce the CPR rate. The discrepancy in cardiopulmonary arrest rates suggests further research should include a nationwide survey to tease out factors involved in in-hospital cardiopulmonary arrest and differences in outcomes based on hospital characteristics.

The Many Hats of Sonic Hedgehog Signaling in Nervous System Development and Disease.

Sonic hedgehog (Shh) signaling occurs concurrently with the many processes that constitute nervous system development. Although Shh is mostly known for its proliferative and morphogenic action through its effects on neural stem cells and progenitors, it also contributes to neuronal differentiation, axonal pathfinding and synapse formation and function. To participate in these diverse events, Shh signaling manifests differently depending on the maturational state of the responsive cell, on the other signaling pathways regulating neural cell function and the environmental cues that surround target cells. Shh signaling is particularly dynamic in the nervous system, ranging from canonical transcription-dependent, to non-canonical and localized to axonal growth cones. Here, we review the variety of Shh functions in the developing nervous system and their consequences for neurodevelopmental diseases and neural regeneration, with particular emphasis on the signaling mechanisms underlying Shh action.

Comparison of intubation times using a manikin with an immobilized cervical spine: Macintosh laryngoscope vs. GlideScope vs. fiberoptic bronchoscope.

OBJECTIVE: Airway management in patients with suspected cervical spine injury is classified as a "difficult airway." The best device for managing difficult airways is not known. Therefore, we conducted an intubation study simulating patients with cervical spine injury using three devices: a conventional Macintosh laryngoscope, a video laryngoscope (GlideScope), and a fiberoptic bronchoscope (MAF-TM). Success rates, intubation time, and complication rates were compared. METHODS: Nine physician experts in airway management participated in this study. Cervical immobilization was used to simulate a difficult airway. Each participant performed intubation using airway devices in a randomly chosen order. We measured the time to vocal cord visualization, time to endotracheal tube insertion, and total tracheal intubation time. Success rates and dental injury rates were compared between devices. RESULTS: Total tracheal intubation time using the Macintosh laryngoscope, GlideScope, and fiberoptic bronchoscope was 13.3 (range, 11.1 to 20.1), 14.9 (range, 12.7 to 22.3), and 19.4 seconds (range, 14.1 to 32.5), respectively. Total tracheal intubation time differed significantly among the devices (P=0.009). Success rates for the Macintosh laryngoscope, GlideScope, and fiberoptic bronchoscope were 98%, 96%, and 100%, respectively, and dental injury rates were 5%, 19%, and 0%, respectively. CONCLUSION: The fiberoptic bronchoscope required longer intubation times than the other devices. However, this device had the best success rate with the least incidence of dental injury.

Spatiotemporal integration of developmental cues in neural development.

Nervous system development relies on the generation of neurons, their differentiation and establishment of synaptic connections. These events exhibit remarkable plasticity and are regulated by many developmental cues. Here, we review the mechanisms of three classes of these cues: morphogenetic proteins, electrical activity, and the environment. We focus on second messenger dynamics and their role as integrators of the action of diverse cues, enabling plasticity in the process of neural development.

A critical role for STIM1 in filopodial calcium entry and axon guidance.

BACKGROUND: Stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum, regulates store-operated Ca2+ entry (SOCE) that is essential for Ca2+ homeostasis in many types of cells. However, if and how STIM1 and SOCE function in nerve growth cones during axon guidance remains to be elucidated. RESULTS: We report that STIM1 and transient receptor potential channel 1 (TRPC1)-dependent SOCE operates in Xenopus spinal growth cones to regulate Ca2+ signaling and guidance responses. We found that STIM1 works together with TRPC1 to mediate SOCE within growth cones and filopodia. In particular, STIM1/TRPC1-dependent SOCE was found to mediate oscillatory filopodial Ca2+ transients in the growth cone. Disruption of STIM1 function abolished filopodial Ca2+ transients and impaired Ca2+-dependent attractive responses of Xenopus growth cones to netrin-1. Finally, interference with STIM1 function was found to disrupt midline axon guidance of commissural interneurons in the developing Xenopus spinal cord in vivo. CONCLUSIONS: Our data demonstrate that STIM1/TRPC1-dependent SOCE plays an essential role in generating spatiotemporal Ca2+ signals that mediate guidance responses of nerve growth cones.

Dynamic localization of G-actin during membrane protrusion in neuronal motility.

BACKGROUND: Actin-based cell motility is fundamental for development, function, and malignant events in eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. RESULTS: Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer-binding proteins profilin and thymosin ß4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. CONCLUSIONS: Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis.

Path integral Monte Carlo with importance sampling for excitons interacting with an arbitrary phonon bath.

The reduced density matrix of excitons coupled to a phonon bath at a finite temperature is studied using the path integral Monte Carlo method. Appropriate choices of estimators and importance sampling schemes are crucial to the performance of the Monte Carlo simulation. We show that by choosing the population-normalized estimator for the reduced density matrix, an efficient and physically-meaningful sampling function can be obtained. In addition, the nonadiabatic phonon probability density is obtained as a byproduct during the sampling procedure. For importance sampling, we adopted the Metropolis-adjusted Langevin algorithm. The analytic expression for the gradient of the target probability density function associated with the population-normalized estimator cannot be obtained in closed form without a matrix power series. An approximated gradient that can be efficiently calculated is explored to achieve better computational scaling and efficiency. Application to a simple one-dimensional model system from the previous literature confirms the correctness of the method developed in this manuscript. The displaced harmonic model system within the single exciton manifold shows the numerically exact temperature dependence of the coherence and population of the excitonic system. The sampling scheme can be applied to an arbitrary anharmonic environment, such as multichromophoric systems embedded in the protein complex. The result of this study is expected to stimulate further development of real time propagation methods that satisfy the detailed balance condition for exciton populations.

Atomistic study of the long-lived quantum coherences in the Fenna-Matthews-Olson complex.

A remarkable amount of theoretical research has been carried out to elucidate the physical origins of the recently observed long-lived quantum coherence in the electronic energy transfer process in biological photosynthetic systems. Although successful in many respects, several widely used descriptions only include an effective treatment of the protein-chromophore interactions. In this work, by combining an all-atom molecular dynamics simulation, time-dependent density functional theory, and open quantum system approaches, we successfully simulate the dynamics of the electronic energy transfer of the Fenna-Matthews-Olson pigment-protein complex. The resulting characteristic beating of populations and quantum coherences is in good agreement with the experimental results and the hierarchy equation of motion approach. The experimental absorption, linear, and circular dichroism spectra and dephasing rates are recovered at two different temperatures. In addition, we provide an extension of our method to include zero-point fluctuations of the vibrational environment. This work thus presents, to our knowledge, one of the first steps to explain the role of excitonic quantum coherence in photosynthetic light-harvesting complexes based on their atomistic and molecular description.

Postsynaptic TRPC1 function contributes to BDNF-induced synaptic potentiation at the developing neuromuscular junction.

Brain-derived neurotrophic factor (BDNF) induces synaptic potentiation at both neuromuscular junctions (NMJs) and synapses of the CNS through a Ca2+ -dependent pathway. The molecular mechanism underlying BDNF-induced synaptic potentiation, especially the regulation of Ca2+ dynamics, is not well understood. Using the Xenopus NMJ in culture as a model system, we show that pharmacological inhibition or morpholino-mediated knockdown of Xenopus TRPC1 (XTRPC1) significantly attenuated the BDNF-induced potentiation of the frequency of spontaneous synaptic responses at the NMJ. Functionally, XTRPC1 was required specifically in postsynaptic myocytes for BDNF-induced Ca2+ elevation and full synaptic potentiation at the NMJ, suggesting a previously underappreciated postsynaptic function of Ca2+ signaling in neurotrophin-induced synaptic plasticity, in addition to its well established role at presynaptic sites. Mechanistically, blockade of the p75 neurotrophin receptor abolished BDNF-induced postsynaptic Ca2+ elevation and restricted BDNF-induced synaptic potentiation, while knockdown of the TrkB receptor in postsynaptic myocytes had no effect. Our study suggests that BDNF-induced synaptic potentiation involves coordinated presynaptic and postsynaptic responses and identifies TRPC1 as a molecular mediator for postsynaptic Ca2+ elevation required for BDNF-induced synaptic plasticity.

Anti-ganglioside antibody-mediated activation of RhoA induces inhibition of neurite outgrowth.

Anti-ganglioside antibodies (Abs) are strongly associated with axonal forms of Guillain Barré syndrome (GBS). Some studies indicate that these Abs, including those with GD1a reactivity, are associated with poor prognosis and/or incomplete recovery. We recently demonstrated that a disease-relevant anti-ganglioside Ab with GD1a reactivity inhibits axon regeneration after PNS injury in an animal model (Lehmann et al., 2007). An implication of these findings is that anti-GD1a Abs can mediate inhibition of axon regeneration and limit recovery in some patients with GBS. The downstream inhibitory intracellular signaling that mediates anti-ganglioside Ab-induced axon inhibition remains unclear. In the current study, we show that disease-relevant and GBS patient's anti-ganglioside Abs can inhibit neurite outgrowth in dissociated primary neuronal cultures. Activation of small GTPase RhoA and its key downstream effector Rho kinase (ROCK) are critical mediators of growth cone and neurite outgrowth inhibition. Therefore, we examined the role of these intracellular signaling molecules in our primary neuronal cultures by molecular and pharmacologic approaches. Our results show that the Ab-mediated inhibition of neurite outgrowth involves the activation of RhoA and ROCK pathway and this activation is through the engagement of specific cell-surface gangliosides by Abs. In summary, these studies directly link patient autoantibodies to an intracellular inhibitory signaling pathway involved in anti-ganglioside Ab-mediated inhibition of neurite outgrowth.

Simplified Sum-Over-States Approach for Predicting Resonance Raman Spectra. Application to Nucleic Acid Bases.

Resonance Raman spectra provide a valuable probe into molecular excited-state structures and properties. Moreover, resonance enhancement is of importance for the chemical contribution to surface-enhanced Raman scattering. In this work, we introduce a simplified sum-over-states scheme for computing Raman spectra and Raman excitation profiles. The proposed sum-over-states approach uses derivatives of electronic excitation energies and transition dipole moments, which can be efficiently computed from time-dependent density functional theory. We analyze and interpret the resonance Raman spectra and Raman excitation profiles of nucleic acid bases using the present approach. Contributions of individual excited states under strictly resonant and nonresonant conditions are investigated, and smooth interpolation between both limiting cases is obtained.

Roles of channels and receptors in the growth cone during PNS axonal regeneration.

Neurons in the peripheral nervous system (PNS) are known to maintain a regenerative capacity and will normally regenerate their axons within a permissive growth environment. The success of regeneration in the PNS largely depends on maintenance of the supportive basal lamina membrane, efficient removal of axonal and myelin debris by macrophages and Schwann cells, expression of neurotrophic factors by Schwann cells, and up-regulation of the intrinsic growth program in PNS neurons. The PNS regenerative process is well characterized through initial Wallerian degeneration followed by axonal sprouting, formation of neuronal growth cones, active axonal growth to the target, and finally sensory and motor functional recovery. The initiation and maintenance of active growth cones during peripheral nerve regeneration recapitulate many aspects of early neural development and are achieved through the activation of complex signaling cascades, involving various receptors, channels, cytoplasmic signaling cascades, as well as transcriptional and translational programs. This review focuses on roles of cell surface ion channels and receptors in the growth cone during Wallerian degeneration and axon regeneration in the PNS.

Peptidyl-prolyl isomerase FKBP52 controls chemotropic guidance of neuronal growth cones via regulation of TRPC1 channel opening.

Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase-independent mechanisms, FKBPs are most highly expressed in the nervous system, where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.

First-principles semiclassical initial value representation molecular dynamics.

In this work, we explore the use of the semiclassical initial value representation (SC-IVR) method with first-principles electronic structure approaches to carry out classical molecular dynamics. The proposed approach can extract the vibrational power spectrum of carbon dioxide from a single trajectory providing numerical results that agree with experiment and quantum calculations. The computational demands of the method are comparable to those of classical single-trajectory calculations, while describing uniquely quantum features such as the zero-point energy and Fermi resonances. The method can also be used to identify symmetry properties of given vibrational peaks and investigate vibrational couplings by selected classical trajectories. The accuracy of the method degrades for the reproduction of anharmonic shifts for high-energy vibrational levels.

A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues.

Neuronal growth cones contain sophisticated molecular machinery precisely regulating their migration in response to complex combinatorial gradients of diverse external cues. The details of this regulation are still largely unknown, in part due to limitations of the currently available experimental techniques. Microfluidic devices have been shown to be capable of generating complex, stable and precisely controlled chemical gradients, but their use in studying growth cone migration has been limited in part due to the effects of shear stress. Here we describe a microfluidics-based turning-assay chip designed to overcome this issue. In addition to generating precise gradients of soluble guidance cues, the chip can also fabricate complex composite gradients of diffusible and surface-bound guidance cues that mimic the conditions the growth cones realistically counter in vivo. Applying this assay to Xenopus embryonic spinal neurons, we demonstrate that the presence of a surface-bound laminin gradient can finely tune the polarity of growth cone responses (repulsion or attraction) to gradients of brain-derived neurotrophic factor (BDNF), with the guidance outcome dependent on the mean BDNF concentration. The flexibility inherent in this assay holds significant potential for refinement of our understanding of nervous system development and regeneration, and can be extended to elucidate other cellular processes involving chemotaxis of shear sensitive cells.