N-Glycosylation Deficiency in Transgene α7 nAChR and RIC3 Expressing CHO Cells Without NACHO.

The human neuronal nicotinic acetylcholine receptor α7 (nAChR) is an important target implicated in diseases like Alzheimer's or Parkinson's, as well as a validated target for drug discovery. For α7 nAChR model systems, correct folding and ion influx functions are essential. Two chaperones, resistance to inhibitors of cholinesterase 3 (RIC3) and novel nAChR regulator (NACHO), enhance the assembly and function of α7 nAChR. This study investigates the consequence of NACHO absence on α7 nAChR expression and function. Therefore, the sequences of human α7 nAChR and human RIC3 were transduced in Chinese hamster ovary (CHO) cells. Protein expression and function of α7 nAChR were confirmed by Western blot and voltage clamp, respectively. Cellular viability was assessed by cell proliferation and lactate dehydrogenase assays. Intracellular and extracellular expression were determined by in/on-cell Western, compared with another nAChR subtype by novel cluster fluorescence-linked immunosorbent assay, and N-glycosylation efficiency was assessed by glycosylation digest. The transgene CHO cell line showed expected protein expression and function for α7 nAChR and cell viability was barely influenced by overexpression. While intracellular levels of α7 nAChR were as anticipated, plasma membrane insertion was low. The glycosylation digest revealed no appreciable N-glycosylation product. This study demonstrates a stable and functional cell line expressing α7 nAChR, whose protein expression, function, and viability are not affected by the absence of NACHO. The reduced plasma membrane insertion of α7 nAChR, combined with incorrect matured N-glycosylation at the Golgi apparatus, suggests a loss of recognition signal for lectin sorting.

Emission Spectroscopy-Based Sensor System to Correlate the In-Cylinder Combustion Temperature of a Diesel Engine to NOx Emissions.

Due to a rising importance of the reduction of pollutant, produced by conventional energy technologies, the knowledge of pollutant forming processes during a combustion is of great interest. In this study the in-cylinder temperature, of a near series diesel engine, is examined with a minimal invasive emission spectroscopy sensor. The soot, nearly a black body radiator, emits light, which is spectrally detected and evaluated with a modified function of Planck's law. The results show a good correlation between the determined temperatures and the NOx concentration, measured in the exhaust gas of the engine, during a variety of engine operating points. A standard deviation between 25 K and 49 K was obtained for the in-cylinder temperature measurements.

Two-color four-wave mixing contribution to an electrostrictive laser-induced grating signal in CO2-N2 mixtures and gas diagnostics: publisher's note.

This publisher's note corrects content in Appl. Opt.62, 8115 (2023)APOPAI0003-693510.1364/AO.497467.

Two-color four-wave mixing contribution to an electrostrictive laser-induced grating signal in CO2-N2 mixtures and gas diagnostics.

Multiparameter determination in the gas phase using the versatile laser-induced grating (LIG) technique is a challenging task due to interdependence of observables on multiple thermodynamic parameters. In C O 2-N 2 mixtures, simultaneous determination of species concentration and gas temperature can be achieved by using an additional C O 2 concentration-dependent contribution to the LIG signal, which appears if 1064 nm pump pulses are employed. This contribution can be attributed to a direct, quasi-resonant two-color four-wave mixing (TCFWM) of the pump and probe radiations in C O 2. A detailed study of the laser power and beam polarization, as well as mixture composition, pressure, and temperature dependencies of the TCFWM intensity relative to that of the LIG signal, allowed for the formulation of analytical relations enabling simultaneous mixture composition and temperature determination.

Laser Applications to Chemical, Security, and Environmental Analysis: introduction to the feature issue.

The eighteenth topical meeting on Laser Applications to Chemical, Security, and Environmental Analysis (LACSEA) was held in Vancouver, Canada from 11-15 July 2022, as part of the Optica Optical Sensors and Sensing Congress in a hybrid format allowing on-site and online attendance. The meeting featured a broad range of distinguished papers focusing on recent advances in laser and optical spectroscopy. A total of 52 contributed and invited papers were presented during the meeting, including topics such as photo-acoustic spectroscopy, imaging, non-linear technologies, frequency combs, remote sensing, environmental monitoring, aerosols, combustion diagnostics, hypersonic flow diagnostics, nuclear diagnostics, fs/ps applications, and machine learning and computational sensing.

Improvement of the coherent model function for S-branch Raman linewidth determination in oxygen.

Determination of S-branch Raman linewidths of oxygen from picosecond time-domain pure rotational coherent anti-Stokes Raman spectroscopy (RCARS) measurements requires consideration of coherence beating. We present an optimization of the established model for fitting the coherence decay in oxygen, which leads to an improvement in Raman linewidth data quality, especially for the challenging small signal intensity and decay constant regime, enabling the application for low oxygen concentrations. Two modifications to the fitting procedure are discussed, which aim at reliably fitting the second coherence beat properly. These are evaluated statistically against simulated decay traces, and weighing the data by the inverse of the data magnitude gives the best agreement. The presented temperature dependent ${{\rm O}_2} {-} {{\rm O}_2}$ S-branch Raman linewidth from the modified model shows an improved data quality over the original model function for all studied temperatures. ${{\rm O}_2} {-} {{\rm N}_2}$ linewidths of oxygen in air for the temperature range from 295 K to 1900 K demonstrate applicability to small concentrations. Use of the determined RCARS ${{\rm O}_2} {-} {{\rm O}_2}$ S-branch linewidth instead of regularly used Q-branch derived linewidths leads to a lowering in evaluated RCARS temperature by about 21 K, thereby, a much better agreement with thermocouple measurements.

Temperature dependent determination of the S-branch Raman linewidths of oxygen and carbon dioxide in an oxyfuel relevant mixture.

The temperature dependence of the ${\rm O}_2$ and ${\rm CO}_2$ S-branch linewidths in a 30/70% ${\rm O}_2 - {\rm CO}_2$ mixture between 295 K and 1900 K has been studied by a picosecond time-resolved pure rotational coherent anti-Stokes Raman scattering (RCARS) approach. The S-branch Raman linewidths are required for diagnostics of thermodynamic properties in oxyfuel combustion processes by RCARS, where this mixture is of special interest, because it is regularly used to replace air when transiting from air-fed to oxyfuel combustion. The obtained linewidths for oxygen and carbon dioxide show a strong deviation from pure self-broadened linewidths and previously used Q-branch linewidths, respectively. A discussion on the expected impact on RCARS thermometry and concentration evaluations as well as a description of specific properties of oxygen and carbon dioxide and their effect on the dephasing behavior of the Raman coherences and, thereby the Raman linewidths, is included, along tabulated linewidths data of both molecules.

Laser applications to chemical, security, and environmental analysis: introduction to the feature issue.

This Applied Optics feature issue on laser applications to chemical, security, and environmental analysis (LACSEA) highlights papers presented at the LACSEA 2020 Seventeenth Topical Meeting sponsored by The Optical Society (OSA).

CH and NO planar laser-induced fluorescence and Rayleigh-scattering in turbulent flames using a multimode optical parametric oscillator.

An optical parametric oscillator (OPO) is developed and characterized for the simultaneous generation of ultraviolet (UV) and near-UV nanosecond laser pulses for the single-shot Rayleigh scattering and planar laser-induced-fluorescence (PLIF) imaging of methylidyne (CH) and nitric oxide (NO) in turbulent flames. The OPO is pumped by a multichannel, 8-pulse Nd:YAG laser cluster that produces up to 225 mJ/pulse at 355 nm with pulse spacing of 100 µs. The pulsed OPO has a conversion efficiency of 9.6% to the signal wavelength of ∼430nm when pumped by the multimode laser. Second harmonic conversion of the signal, with 3.8% efficiency, is used for the electronic excitation of the A-X (1,0) band of NO at ∼215nm, while the residual signal at 430 nm is used for direct excitation of the A-X (0,0) band of the CH radical and elastic Rayleigh scattering. The section of the OPO signal wavelength for simultaneous CH and NO PLIF imaging is performed with consideration of the pulse energy, interference from the reactant and product species, and the fluorescence signal intensity. The excitation wavelengths of 430.7 nm and 215.35 nm are studied in a laminar, premixed CH4-H2-NH3-air flame. Single-shot CH and NO PLIF and Rayleigh scatter imaging is demonstrated in a turbulent CH4-H2-NH3 diffusion flame using a high-speed intensified CMOS camera. Analysis of the complementary Rayleigh scattering and CH and NO PLIF enables identification and quantification of the high-temperature flame layers, the combustion product zones, and the fuel-jet core. Considerations for extension to simultaneous, 10-kHz-rate acquisition are discussed.

Determination of N2-N2 and N2-O2 S-branch Raman linewidths using time-resolved picosecond pure rotational coherent anti-Stokes Raman scattering.

N2-N2 and N2-O2 S-branch Raman linewidths have been determined using picosecond dual-broadband pure rotational coherent anti-Stokes Raman scattering (CARS). Time-resolved rotational CARS measurements were performed in gas-phase N2 and air for temperatures up to 1900 K in order to determine the time constants of the coherence decay to subsequently calculate the S-branch Raman linewidths. Coherence decay time traces and the resulting S-branch Raman linewidths are presented for N2-N2 and N2-O2 collisions. Therewith, we reduce the gap of widely missing S-branch linewidth data in the temperature regime of many combustion processes. Further, we demonstrate that the standard monoexponential fitting of the coherence decay, as it is commonly done for nitrogen, is not applicable to oxygen.

Laser applications to chemical, security, and environmental analysis: introduction to the feature issue.

This Applied Optics feature issue on laser applications to chemical, security, and environmental analysis (LACSEA) highlights papers presented at the LACSEA 2018 Sixteenth Topical Meeting sponsored by the Optical Society of America.

Immediate responses of the cockroach Blaptica dubia after the exposure to sulfur mustard.

The chemical agent sulfur mustard (SM) causes erythema, skin blisters, ulcerations, and delayed wound healing. It is accepted that the underlying molecular toxicology is based on DNA alkylation. With an expected delay, DNA damage causes impairment of protein biosynthesis and disturbance of cell division. However, using the cockroach model Blaptica dubia, the presented results show that alkylating compounds provoke immediate behavior responses along with fast changes in the electrical field potential (EFP) of neurons, suggesting that lesions of DNA are probably not the only effect of alkylating compounds. Blaptica dubia was challenged with SM or 2-chloroethyl-ethyl sulfide (CEES). Acute toxicity was objectified by a disability score. Physiological behavior responses (antennae pullback reflex, escape attempts, and grooming) were monitored after exposure. To estimate the impact of alkylating agents on neuronal activity, EFP recordings of the antennae and the thoracic ganglion were performed. After contact to neat SM, a pullback reflex of the antennae was the first observation. Subsequently, a striking escape behavior occured which was characterized by persistent movement of the legs. In addition, an instantaneous processing of the electrical firing pattern from the antennae to the descending ganglia was detectable. Remarkably, comparing the toxicity of the applied alkylating agents, effects induced by CEES were much more pronounced compared to SM. In summary, our findings document immediate effects of B. dubia after exposure to alkylating substances. These fast responses cannot be interpreted as a consequence of DNA alkylation. Therefore, the dogma that DNA alkylation is the exclusive cause for SM toxicity has to be questioned.

Laser applications to chemical, security, and environmental analysis: introduction to the feature issue.

This Applied Optics feature issue on laser applications to chemical, security, and environmental analysis (LACSEA) highlights papers presented at the LACSEA 2016 Fifteenth Topical Meeting sponsored by the Optical Society of America.

Three-color vibrational CARS thermometry of fuel-rich ethylene/air flames using a potassium gadolinium tungstate Raman-active crystal as a source of narrowband probe radiation.

Three-color broadband vibrational coherent anti-Stokes Raman scattering (CARS) temperature measurements were carried out in laminar fuel-rich sooting ethylene/air flames. Stimulated Raman scattering (SRS) of a picosecond pump laser pulse in a Raman-active potassium gadolinium tungstate [KGd(WO4)2] crystal was employed as a source of narrowband probe radiation. In the three-color CARS experiment, this wavelength-shifted radiation enables N2-based vibrational CARS temperature measurements in sooting flames free of the signal interference with the absorption/emission bands of the flame intermediate radicals C2. Spatial temperature profiles for different fuel-rich atmospheric pressure ethylene/air flames are presented in comparison with the results of two-color broadband vibrational and dual-broadband pure rotational CARS temperature measurements. The comparison shows the suitability of the three-color CARS measurement technique employing the KGd(WO4)2 crystal for accurate, C2 interference-free, temperature measurements in sooting flames.

Efficacy of the rePON1 mutant IIG1 to prevent cyclosarin toxicity in vivo and to detoxify structurally different nerve agents in vitro.

The potent human toxicity of organophosphorus (OP) nerve agents calls for the development of effective antidotes. Standard treatment for nerve agent poisoning with atropine and an oxime has a limited efficacy. An alternative approach is the development of catalytic bioscavengers using OP-hydrolyzing enzymes such as paraoxonases (PON1). Recently, a chimeric PON1 mutant, IIG1, was engineered toward the hydrolysis of the toxic isomers of soman and cyclosarin with high in vitro catalytic efficiency. In order to investigate the suitability of IIG1 as a catalytic bioscavenger, an in vivo guinea pig model was established to determine the protective effect of IIG1 against the highly toxic nerve agent cyclosarin. Prophylactic i.v. injection of IIG1 (1 mg/kg) prevented systemic toxicity in cyclosarin (~2LD50)-poisoned guinea pigs, preserved brain acetylcholinesterase (AChE) activity, and protected erythrocyte AChE activity partially. A lower IIG1 dose (0.2 mg/kg) already prevented mortality and reduced systemic toxicity. IIG1 exhibited a high catalytic efficiency with a homologous series of alkylmethylfluorophosphonates but had low efficiency with the phosphoramidate tabun and was virtually ineffective with the nerve agent VX. This quantitative analysis validated the model for predicting in vivo protection by catalytic bioscavengers based on their catalytic efficiency, the level of circulating enzyme, and the dose of the intoxicating nerve agent. The in vitro and in vivo results indicate that IIG1 may be considered as a promising candidate bioscavenger to protect against the toxic effects of a range of highly toxic nerve agents.

Cell-Sonar, a Novel Method for Intracellular Tracking of Secretory Pathways.

BACKGROUND: Intracellular tracking is commonly used in trafficking research. Until today, the respective techniques have remained complex, and complicated, mostly transgenic target protein changes are necessary, often requiring expensive equipment and expert knowledge. METHODS: We present a novel method, which we term "cell-sonar", that enables the user to track expression changes of specific protein markers that serve as points of interaction. Our study provides comparable analyses of expression changes of these marker proteins by in-cell Western analyses in two otherwise isogenic cell lines that only differ in the overexpression of the tracked target protein. Using the overexpressed human adult muscle-type nicotinic acetylcholine receptor as an example, we demonstrate that cell-sonar can cover multiple intracellular compartments such as the endoplasmic reticulum, the pathway between it and the Golgi apparatus, and the endocytic pathway. RESULTS: We provide evidence for receptor maturation in the Golgi and storage in recycling endosomes, rather than the fate of increased insertion into the plasma membrane. Additionally, we demonstrate with the implementation of nicotine that the receptor's destiny is exasperated up to secondary degradation. CONCLUSIONS: Cell-sonar is an affordable, easy-to-implement, and cheap method that can be adapted to a broad variety of proteins and cellular pathways of interest to researchers.

Restoration of nerve agent impaired neuromuscular transmission in rat diaphragm by bispyridinium non-oximes - Structure-activity relationships.

Organophosphate (OP) poisoning is currently treated with atropine, oximes and benzodiazepines. The nicotinic signs, i.e., respiratory impairment, can only be targeted indirectly via the use of oximes as reactivators of OP-inhibited acetylcholinesterase. Hence, compounds selectively targeting nicotinic acetylcholine receptors (nAChRs) might fundamentally improve current treatment options. The bispyridinium compound MB327 has previously shown some therapeutic effect against nerve agents in vitro and in vivo. Nevertheless, compound optimization was deemed necessary, due to limitations (e.g., toxicity and efficacy). The current study investigated a series of 4-tert-butyl bispyridinium compounds and of corresponding bispyridinium compounds without substituents in a rat diaphragm model using an indirect field stimulation technique. The length of the respective linker influenced the ability of the bispyridinium compounds to restore muscle function in rat hemidiaphragms. The current data show structure-activity relationships for a series of bispyridinium compounds and provide insight for future structure-based molecular modeling.

The use of bispyridinium non-oxime analogues for the restoration of nerve agent impaired neuromuscular transmission in rat hemidiaphragms - Structure optimization.

Organophosphate pesticide poisoning challenges health care systems worldwide. Furthermore, nerve agents remain a continuous threat. The treatment options for organophosphate poisoning have virtually been unchanged for decades, relying on symptomatic treatment and the use of oximes to indirectly restore neuromuscular function. Hence, compounds targeting directly nicotinic acetylcholine receptors (nAChRs) might substantially improve treatment options. The current study investigated a series of bispyridinium analogues with a trimethylene or 2,2'-diethyloxy linker in a rat hemidiaphragm model, using indirect field stimulation. Methyl- and ethyl-substituted bispyridinium analogues restored neuromuscular function up to 37 ± 17% (MB419, a 3-methyl analogue) at a stimulation frequency of 20 Hz. The bispyridinium analogues with a 2- or 3-methyl group, or a 2- or 3-ethyl group, tended towards a higher restoration of neuromuscular function than those with a 4-methyl or 4-ethyl group, respectively. The current data can be used for future studies to optimize structure-based molecular modeling of compounds targeting the nAChR.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor.

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Screening for new ligands of the MB327-PAM-1 binding site of the nicotinic acetylcholine receptor.

Intoxications with organophosphorus compounds (OPCs) effect a severe impairment of cholinergic neurotransmission that, as a result of overstimulation may lead to desensitization of nicotinic acetylcholine receptors (nAChRs) and finally to death due to respiratory paralysis. So far, therapeutics, that are capable to address and revert desensitized neuromuscular nAChRs into their resting, i.e. functional state are still missing. Still, among a class of compounds termed bispyridinium salts, which are characterized by the presence of two pyridinium subunits, constituents have been identified, that can counteract organophosphate poisoning by resensitizing desensitized nAChRs. According to comprehensive modeling studies this effect is mediated by an allosteric binding site at the nAChR termed MB327-PAM-1 site. For MB327, the most prominent representative of the bispyridinium salts and all other analogues studied so far, the affinity for the aforementioned binding site and the intrinsic activity measured in ex vivo and in in vivo experiments are distinctly too low, to meet the criteria to be fulfilled for therapeutic use. Hence, in order to identify new compounds with higher affinities for the MB327-PAM-1 binding site, as a basic requirement for an enhanced potency, two compound libraries, the ChemDiv library with 60 constituents and the Tocriscreen Plus library with 1280 members have been screened for hit compounds addressing the MB327-PAM-1 binding site, utilizing the [2H6]MB327 MS Binding Assay recently developed by us. This led to the identification of a set of 10 chemically diverse compounds, all of which exhibit an IC50 value of ≤ 10 µM (in the [2H6]MB327 MS Binding Assay), which had been defined as selection criteria. The three most affine ligands, which besides a quinazoline scaffold share similarities with regard to the substitution pattern and the nature of the substituents, are UNC0638, UNC0642 and UNC0646. With binding affinities expressed as pKi values of 6.01 ± 0.10, 5.97 ± 0.05 and 6.23 ± 0.02, respectively, these compounds exceed the binding affinity of MB327 by more than one log unit. This renders them promising starting points for the development of drugs for the treatment of organophosphorus poisoning by addressing the MB327-PAM-1 binding site of the nAChR.