An Investigation of Lipid Modulation of Low Voltage Activated Currents in Spiral Ganglion Neurons

Type I spiral ganglion neurons (SGNs) synapse onto cochlear inner hair cells and constitute the majority of afferent fibres in the auditory nerve (AN). Better characterisation of their biophysical properties may identify therapeutic targets for optimising AN sensitivity. This study aimed to characterise the membrane physiology underlying the firing properties of post-hearing onset SGNs and investigated whether their properties could be modified by the presence of native and synthetic lipids. In dissociated ganglionic cultures, SGNs displayed an intrinsic variation in their firing properties; this could be correlated with the magnitudes of specific membrane currents. SGNs were categorised by their response to depolarising current injection; SGNs either adapted to the stimulus rapidly, slowly or not at all. Rapid adaptation, a mechanism that preserves temporal precision throughout the auditory system, was found to be regulated by a dendrotoxin-K (DTX-K) and tityustoxin-Kα (TsTx)-sensitive low-threshold voltage-activated (LVA) K+ current, suggesting contribution by Kv1.1 and Kv1.2 subunits. As Kv1.2 channels were known to be positively modulated by membrane phosphoinositides, we investigated the influence of phosphatidylinositol-4,5- bisphosphate (PIP2) availability on SGN K+ currents. Inhibiting PIP2 production using wortmannin, or sequestration using a palmitoylated peptide (PIP2-PP), slowed or abolished adaptation in SGNs. PIP2-PP specifically reduced SGN LVA currents in a manner that was partly rescued by intracellular dialysis with diC8PIP2, a nonhydrolysable analogue of PIP2. PIP2-PP application induced similar levels of current inhibition in Kv1.1/Kv1.2 channels heterologously expressed in HEK293 cells. Accordingly, the lipid sensitivity of the Kv1.2 channel was further explored with a range of native and synthetic free fatty acids. Polyunsaturated fatty acids were found to be strong inhibitors of Kv1.2 currents, offering further potential candidates for SGN modulation. Collectively, this data identifies Kv1.1 and Kv1.2 containing K+ channels as key regulators of excitability in the AN, and potential targets for pharmacological modulation

Continuous flow processing as a tool for the generation of terpene-derived monomer libraries

We report the development of a continuous flow approach for the preparation of two bio-derived monomer libraries. A small range of terpenes (ocimene, myrcene, α-terpinene, α-phellandrene, isoprene, and farnesene) have been used as the base set for the library, with the first library derived from a Diels–Alder reaction with the platform chemical maleic anhydride. The second library requires the derivatization of the first through a hydrogenation reaction. The potential for scale-up of both libraries has been demonstrated, with the Diels–Alder process delivering 10.5 grams of the product in 3 hours and the hydrogenation process delivering 10 grams of the material in 16 hours

Quantum memory for entangled two-mode squeezed states

A quantum memory for light is a key element for the realization of future quantum information networks. Requirements for a good quantum memory are (i) versatility (allowing a wide range of inputs) and (ii) true quantum coherence (preserving quantum information). Here we demonstrate such a quantum memory for states possessing Einstein-Podolsky-Rosen (EPR) entanglement. These multi-photon states are two-mode squeezed by 6.0 dB with a variable orientation of squeezing and displaced by a few vacuum units. This range encompasses typical input alphabets for a continuous variable quantum information protocol. The memory consists of two cells, one for each mode, filled with cesium atoms at room temperature with a memory time of about 1msec. The preservation of quantum coherence is rigorously proven by showing that the experimental memory fidelity 0.52(2) significantly exceeds the benchmark of 0.45 for the best possible classical memory for a range of displacements.Comment: main text 5 pages, supplementary information 3 page

PABPN1 gene therapy for oculopharyngeal muscular dystrophy

International audienceOculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by ptosis, swallowing difficulties, proximal limb weakness and nuclear aggregates in skeletal muscles. OPMD is caused by a trinucleotide repeat expansion in the PABPN1 gene that results in an N-terminal expanded polyalanine tract in polyA-binding protein nuclear 1 (PABPN1). Here we show that the treatment of a mouse model of OPMD with an adeno-associated virus-based gene therapy combining complete knockdown of endogenous PABPN1 and its replacement by a wild-type PABPN1 substantially reduces the amount of insoluble aggregates, decreases muscle fibrosis, reverts muscle strength to the level of healthy muscles and normalizes the muscle transcriptome. The efficacy of the combined treatment is further confirmed in cells derived from OPMD patients. These results pave the way towards a gene replacement approach for OPMD treatment

Comparing aerosol number and mass exhalation rates from children and adults during breathing, speaking and singing

Aerosol particles of respirable size are exhaled when individuals breathe, speak and sing and can transmit respiratory pathogens between infected and susceptible individuals. The COVID-19 pandemic has brought into focus the need to improve the quantification of the particle number and mass exhalation rates as one route to provide estimates of viral shedding and the potential risk of transmission of viruses. Most previous studies have reported the number and mass concentrations of aerosol particles in an exhaled plume. We provide a robust assessment of the absolute particle number and mass exhalation rates from measurements of minute ventilation using a non-invasive Vyntus Hans Rudolf mask kit with straps housing a rotating vane spirometer along with measurements of the exhaled particle number concentrations and size distributions. Specifically, we report comparisons of the number and mass exhalation rates for children (12–14 years old) and adults (19–72 years old) when breathing, speaking and singing, which indicate that child and adult cohorts generate similar amounts of aerosol when performing the same activity. Mass exhalation rates are typically 0.002–0.02 ng s−1 from breathing, 0.07–0.2 ng s−1 from speaking (at 70–80 dBA) and 0.1–0.7 ng s−1 from singing (at 70–80 dBA). The aerosol exhalation rate increases with increasing sound volume for both children and adults when both speaking and singing

Profiling quality of care: Is there a role for peer review?

BACKGROUND: We sought to develop a more reliable structured implicit chart review instrument for use in assessing the quality of care for chronic disease and to examine if ratings are more reliable for conditions in which the evidence base for practice is more developed. METHODS: We conducted a reliability study in a cohort with patient records including both outpatient and inpatient care as the objects of measurement. We developed a structured implicit review instrument to assess the quality of care over one year of treatment. 12 reviewers conducted a total of 496 reviews of 70 patient records selected from 26 VA clinical sites in two regions of the country. Each patient had between one and four conditions specified as having a highly developed evidence base (diabetes and hypertension) or a less developed evidence base (chronic obstructive pulmonary disease or a collection of acute conditions). Multilevel analysis that accounts for the nested and cross-classified structure of the data was used to estimate the signal and noise components of the measurement of quality and the reliability of implicit review. RESULTS: For COPD and a collection of acute conditions the reliability of a single physician review was quite low (intra-class correlation = 0.16–0.26) but comparable to most previously published estimates for the use of this method in inpatient settings. However, for diabetes and hypertension the reliability is significantly higher at 0.46. The higher reliability is a result of the reviewers collectively being able to distinguish more differences in the quality of care between patients (p < 0.007) and not due to less random noise or individual reviewer bias in the measurement. For these conditions the level of true quality (i.e. the rating of quality of care that would result from the full population of physician reviewers reviewing a record) varied from poor to good across patients. CONCLUSIONS: For conditions with a well-developed quality of care evidence base, such as hypertension and diabetes, a single structured implicit review to assess the quality of care over a period of time is moderately reliable. This method could be a reasonable complement or alternative to explicit indicator approaches for assessing and comparing quality of care. Structured implicit review, like explicit quality measures, must be used more cautiously for illnesses for which the evidence base is less well developed, such as COPD and acute, short-course illnesses

Heterologous Expression and Patch-Clamp Recording of P2X Receptors in HEK293 Cells

P2X receptors (P2XRs) are ligand-gated ion channels gated by extracellular adenosine 5′-triphosphate (ATP) and play a critical role in mediating ATP-induced purinergic signaling in physiological and pathological processes. Heterologous expression of P2XR in human embryonic kidney 293 (HEK293) cells and measurement of P2XR-mediated currents using patch-clamp recording technique have been widely used to study the biophysical and pharmacological properties of these receptors. Combination of electrophysiology with site-directed mutagenesis and structural information has shed light on the molecular basis for receptor activation and mechanisms of actions by receptor antagonists and modulators. It is anticipated that such methodologies will continue helping us to provide more mechanistic understanding of P2XRs and to test novel receptor antagonists and allosteric modulators for therapeutical purposes. In this chapter, we describe protocols of transiently or stably expressing the P2XR in HEK293 cells and measuring P2XR-mediated currents by using whole-cell recording

Functional Characterization of the HuR:CD83 mRNA Interaction

Maturation of dendritic cells (DC) is characterized by expression of CD83, a surface protein that appears to be necessary for the effective activation of naïve T-cells and T-helper cells by DC. Lately it was shown that CD83 expression is regulated on the posttranscriptional level by interaction of the shuttle protein HuR with a novel posttranscriptional regulatory RNA element (PRE), which is located in the coding region of the CD83 transcript. Interestingly, this interaction commits the CD83 mRNA to efficient nuclear export via the CRM1 pathway. To date, however, the structural basis of this interaction, which potentially involves three distinct RNA recognition motifs (RRM1–3) in HuR and a complex three-pronged RNA stem-loop element in CD83 mRNA, has not been investigated in detail. In the present work we analyzed this interaction in vitro and in vivo using various HuR- and CD83 mRNA mutants. We are able to demonstrate that both, RRM1 and RRM2 are crucial for binding, whereas RRM3 as well as the HuR hinge region contributed only marginally to this protein∶RNA interaction. Furthermore, mutation of uridine rich patches within the PRE did not disturb HuR:CD83 mRNA complex formation while, in contrast, the deletion of specific PRE subfragments from the CD83 mRNA prevented HuR binding in vitro and in vivo. Interestingly, the observed inhibition of HuR binding to CD83 mRNA does not lead to a nuclear trapping of the transcript but rather redirected this transcript from the CRM1- towards the NXF1/TAP-specific nuclear export pathway. Thus, the presence of a functional PRE permits nucleocytoplasmic trafficking of the CD83 transcript via the CRM1 pathway

Spontaneous Breathing in Early Acute Respiratory Distress Syndrome: Insights From the Large Observational Study to UNderstand the Global Impact of Severe Acute Respiratory FailurE Study

OBJECTIVES: To describe the characteristics and outcomes of patients with acute respiratory distress syndrome with or without spontaneous breathing and to investigate whether the effects of spontaneous breathing on outcome depend on acute respiratory distress syndrome severity. DESIGN: Planned secondary analysis of a prospective, observational, multicentre cohort study. SETTING: International sample of 459 ICUs from 50 countries. PATIENTS: Patients with acute respiratory distress syndrome and at least 2 days of invasive mechanical ventilation and available data for the mode of mechanical ventilation and respiratory rate for the 2 first days. INTERVENTIONS: Analysis of patients with and without spontaneous breathing, defined by the mode of mechanical ventilation and by actual respiratory rate compared with set respiratory rate during the first 48 hours of mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Spontaneous breathing was present in 67% of patients with mild acute respiratory distress syndrome, 58% of patients with moderate acute respiratory distress syndrome, and 46% of patients with severe acute respiratory distress syndrome. Patients with spontaneous breathing were older and had lower acute respiratory distress syndrome severity, Sequential Organ Failure Assessment scores, ICU and hospital mortality, and were less likely to be diagnosed with acute respiratory distress syndrome by clinicians. In adjusted analysis, spontaneous breathing during the first 2 days was not associated with an effect on ICU or hospital mortality (33% vs 37%; odds ratio, 1.18 [0.92-1.51]; p = 0.19 and 37% vs 41%; odds ratio, 1.18 [0.93-1.50]; p = 0.196, respectively ). Spontaneous breathing was associated with increased ventilator-free days (13 [0-22] vs 8 [0-20]; p = 0.014) and shorter duration of ICU stay (11 [6-20] vs 12 [7-22]; p = 0.04). CONCLUSIONS: Spontaneous breathing is common in patients with acute respiratory distress syndrome during the first 48 hours of mechanical ventilation. Spontaneous breathing is not associated with worse outcomes and may hasten liberation from the ventilator and from ICU. Although these results support the use of spontaneous breathing in patients with acute respiratory distress syndrome independent of acute respiratory distress syndrome severity, the use of controlled ventilation indicates a bias toward use in patients with higher disease severity. In addition, because the lack of reliable data on inspiratory effort in our study, prospective studies incorporating the magnitude of inspiratory effort and adjusting for all potential severity confounders are required