The role of Nav1.9 channel in the development of neuropathic orofacial pain associated with trigeminal neuralgia

BACKGROUND: Trigeminal neuralgia is accompanied by severe mechanical, thermal and chemical hypersensitivity of the orofacial area innervated by neurons of trigeminal ganglion (TG). We examined the role of the voltage-gated sodium channel subtype Nav1.9 in the development of trigeminal neuralgia. RESULTS: We found that Nav1.9 is required for the development of both thermal and mechanical hypersensitivity induced by constriction of the infraorbital nerve (CION). The CION model does not induce change on Nav1.9 mRNA expression in the ipsilateral TG neurons when evaluated 9 days after surgery. CONCLUSIONS: These results demonstrate that Nav1.9 channels play a critical role in the development of orofacial neuropathic pain. New routes for the treatment of orofacial neuropathic pain focussing on regulation of the voltage-gated Nav1.9 sodium channel activity should be investigated

Optimal planning and campaign scheduling of biopharmaceutical processes using a continuous-time formulation

This work addresses the optimal planning and campaign scheduling of biopharmaceutical manufacturing processes, considering multiple operational characteristics, such as the campaign schedule of batch and/or continuous process steps, multiple intermediate deliveries, sequence dependent changeovers operations, product storage restricted to shelf-life limitations, and the track-control of the production/campaign lots due to regulatory policies. A new mixed integer linear programing (MILP) model, based on a Resource Task Network (RTN) continuous time single-grid formulation, is developed to comprise the integration of all these features. The performance of the model features is discussed with the resolution of a set of industrial problems with different data sets and process layouts, demonstrating the wide application of the proposed formulation. It is also performed a comparison with a related literature model, showing the advantages of the continuous-time approach and the generality of our model for the optimal production management of biopharmaceutical processes

Translating chitosan to clinical delivery of nucleic acid-based drugs

A number of systems based on synthetic molecules, among them cationic liposomes and poly(ethylene imine)-based polymers, have been proposed as delivery vehicles for nucleic acids. Some of these systems have even reached the market, ensuring efficient and transient transfection levels in a variety of cell types. However, toxicity issues have limited their application in vivo. In this context, chitosan, a biocompatible and biodegradable polysaccharide, has been proposed as a promising alternative for the delivery of nucleic acid-based molecules. Here we present an overview of the state of the art of chitosan-based vectors for nucleic acid delivery and the most recent data on the in vivo testing of the proposed systems. We additionally express our view on the barriers that might be hampering the translation of this knowledge into clinical practice and the challenges that need to be fulfilled for these promising vehicles to reach patients.The Programa Operacional Factores de Competitividade — COMPETE and the Portuguese funds through FCT— Fundação para a Ciência e a Tecnologia (PTDC/CTM-NAN/115124/2009 and PEst C/SAU/LA0002/2011) that supported this work. C.P.G. and C.D.F.L. acknowledge FCT for their PhD scholarships (SFRH/BD/79930/2011 and SFRH/BD/77933/2011). P.M.D.M. is supported by a Marie Curie Actions grant within the framework of the European Union’s Seventh Framework Program (PIEF-GA-2011–300485). The authors would like to thank A. Nunes (IBMC-INEB) for her contribution to the graphic design of Figure 2

Sensory neuron–derived NaV1.7 contributes to dorsal horn neuron excitability

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno–electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system–specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis

Tools for analysis and conditional deletion of subsets of sensory neurons [version 1; peer review: 4 approved]

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. // Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. // Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. // Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders

A high-throughput bioimaging study to assess the impact of chitosan-based nanoparticle degradation on DNA delivery performance

By using imaging flow cytometry as a powerful statistical high-throughput technique we investigated the impact of degradation on the biological performance of trimethyl chitosan (TMC)-based nanoparticles (NPs). In order to achieve high transfection efficiencies, a precise balance between NP stability and degradation must occur. We altered the biodegradation rate of the TMC NPs by varying the degree of acetylation (DA) of the polymer (DA ranged from 4 to 21%), giving rise to NPs with different enzymatic degradation profiles. While this parameter did not affect NP size, charge or ability to protect plasmid DNA, NPs based on TMC with an intermediate DA (16%) showed the highest transfection efficiency. Subsequently, by means of a single quantitative technique, we were able to follow, for each tested formulation, major steps of the NP-mediated gene delivery process – NP cell membrane association, internalization and intracellular trafficking, including plasmid DNA transport towards the nucleus. NP cytotoxicity was also possible to determine by quantification of cell apoptosis. Overall, the obtained data revealed that the biodegradation rate of these NPs affects their intracellular trafficking and, consequently, their efficiency to transfect cells. Thus, one can use the polymer DA to modulate the NPs towards attaining different degradation rates and tune their bioactivity according to the desired application. Furthermore, this novel technical approach revealed to be a valuable tool for the initial steps of nucleic acid vector design. Statement of Significance By changing the biodegradation rate of trimethyl chitosan-based nanoparticles (NPs) one was able to alter the NP ability to protect or efficiently release DNA and consequently, to modulate their intracellular dynamics. To address the influence of NP degradation rate in their transfection efficiency we took advantage of imaging flow cytometry, a high-throughput bioimaging technique, to unravel some critical aspects about NP formulation such as the distinction between internalized versus cell-associated/adsorbed NP, and even explore NP intracellular localization. Overall, our work provides novel information about the importance of vector degradation rate for gene delivery into cells, as a way to tune gene expression as a function of the desired application, and advances novel approaches to optimize nanoparticle formulation.The work was financed by Portuguese funds through FCT (Fundação para a Ciência e a Tecnologia) in the framework of the projects UID/BIM/04293/2013, PTDC/CTM-NAN/115124/2009 and PTDC/CTM-NAN/3547/2014. CP Gomes, CDF Lopes and PMD Moreno acknowledge FCT for their scholarships (SFRH/BD/79930/2011, SFRH/BD/77933/2011 and SFRH/BPD/108738/2015, respectively). Authors acknowledge the Bioimaging Center for Biomaterials and Regenerative Therapies of INEB (b.IMAGE) for the support with Imaging Flow Cytometry and the Biointerfaces and Nanotechnology Service (INEB-i3S) for size and zeta potential analysis

Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based Nanoparticles

Splice switching oligonucleotides (SSOs) are a class of single-stranded antisense oligonucleotides (ssONs) being used as gene therapeutics and demonstrating great therapeutic potential. The availability of biodegradable and biocompatible delivery vectors that could improve delivery efficiencies, reduce dosage, and, in parallel, reduce toxicity concerns could be advantageous for clinical translation. In this work we explored the use of quaternized amphiphilic chitosan-based vectors in nanocomplex formation and delivery of splice switching oligonucleotides (SSO) into cells, while providing insights regarding cellular uptake of such complexes. Results show that the chitosan amphiphilic character is important when dealing with SSOs, greatly improving colloidal stability under serum conditions, as analyzed by dynamic light scattering, and enhancing cellular association. Nanocomplexes were found to follow an endolysosomal route with a long lysosome residence time. Conjugation of a hydrophobic moiety, stearic acid, to quaternized chitosan was a necessary condition to achieve transfection, as an unmodified quaternary chitosan was completely ineffective. We thus demonstrate that amphiphilic quaternized chitosan is a biomaterial that holds promise and warrants further development as a platform for SSO delivery strategies.This work was cofinanced by Fundacão para a Ciência e a Tecnologia (FCT, Portugal) within projects HMSP-ICT/0020/2010 and PTDC/CTM-NAN/NAN/115124/2009. Additionally, P.M.D.M.acknowledges the support from the Marie Curie Actions of the European Community’s 7th Framework Program (PIEF-GA-2011-300485); J.C.S. acknowledges the graduate fellowship from Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq, Ministry of Science and Technology, Brazil); C.P.G. and V.L. acknowledge FCT for their scholarships (SFRH/BD/79930/2011 and SFRH/BPD/69110/2010). We thank M. Lázaro from the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE) for help with confocal microscopy. 1H NMR and Cryo-SEM were performed at the Centro de Materiais daUniversidade do Porto (CEMUP)

Sonic Hedgehog Is a Chemoattractant for Midbrain Dopaminergic Axons

Midbrain dopaminergic axons project from the substantia nigra (SN) and the ventral tegmental area (VTA) to rostral target tissues, including the striatum, pallidum, and hypothalamus. The axons from the medially located VTA project primarily to more medial target tissues in the forebrain, whereas the more lateral SN axons project to lateral targets including the dorsolateral striatum. This structural diversity underlies the distinct functions of these pathways. Although a number of guidance cues have been implicated in the formation of the distinct axonal projections of the SN and VTA, the molecular basis of their diversity remains unclear. Here we investigate the molecular basis of structural diversity in mDN axonal projections. We find that Sonic Hedgehog (Shh) is expressed at a choice point in the course of the rostral dopaminergic projections. Furthermore, in midbrain explants, dopaminergic projections are attracted to a Shh source. Finally, in mice in which Shh signaling is inactivated during late neuronal development, the most medial dopaminergic projections are deficient

Neural Synchrony during Response Production and Inhibition

Inhibition of irrelevant information (conflict monitoring) and/or of prepotent actions is an essential component of adaptive self-organized behavior. Neural dynamics underlying these functions has been studied in humans using event-related brain potentials (ERPs) elicited in Go/NoGo tasks that require a speeded motor response to the Go stimuli and withholding a prepotent response when a NoGo stimulus is presented. However, averaged ERP waveforms provide only limited information about the neuronal mechanisms underlying stimulus processing, motor preparation, and response production or inhibition. In this study, we examine the cortical representation of conflict monitoring and response inhibition using time-frequency analysis of electroencephalographic (EEG) recordings during continuous performance Go/NoGo task in 50 young adult females. We hypothesized that response inhibition would be associated with a transient boost in both temporal and spatial synchronization of prefrontal cortical activity, consistent with the role of the anterior cingulate and lateral prefrontal cortices in cognitive control. Overall, phase synchronization across trials measured by Phase Locking Index and phase synchronization between electrode sites measured by Phase Coherence were the highest in the Go and NoGo conditions, intermediate in the Warning condition, and the lowest under Neutral condition. The NoGo condition was characterized by significantly higher fronto-central synchronization in the 300–600 ms window, whereas in the Go condition, delta- and theta-band synchronization was higher in centro-parietal regions in the first 300 ms after the stimulus onset. The present findings suggest that response production and inhibition is supported by dynamic functional networks characterized by distinct patterns of temporal and spatial synchronization of brain oscillations

Performance of CMS muon reconstruction in pp collision events at sqrt(s) = 7 TeV

The performance of muon reconstruction, identification, and triggering in CMS has been studied using 40 inverse picobarns of data collected in pp collisions at sqrt(s) = 7 TeV at the LHC in 2010. A few benchmark sets of selection criteria covering a wide range of physics analysis needs have been examined. For all considered selections, the efficiency to reconstruct and identify a muon with a transverse momentum pT larger than a few GeV is above 95% over the whole region of pseudorapidity covered by the CMS muon system, abs(eta) < 2.4, while the probability to misidentify a hadron as a muon is well below 1%. The efficiency to trigger on single muons with pT above a few GeV is higher than 90% over the full eta range, and typically substantially better. The overall momentum scale is measured to a precision of 0.2% with muons from Z decays. The transverse momentum resolution varies from 1% to 6% depending on pseudorapidity for muons with pT below 100 GeV and, using cosmic rays, it is shown to be better than 10% in the central region up to pT = 1 TeV. Observed distributions of all quantities are well reproduced by the Monte Carlo simulation.Comment: Replaced with published version. Added journal reference and DO