Optimisation of a Microwave Synthesis of Silver Nanoparticles by a Quality by Design Approach to Improve SERS Analytical Performances.

A major limitation preventing the use of surface-enhanced Raman scattering (SERS) in routine analyses is the signal variability due to the heterogeneity of metallic nanoparticles used as SERS substrates. This study aimed to robustly optimise a synthesis process of silver nanoparticles to improve the measured SERS signal repeatability and the protocol synthesis repeatability. The process is inspired by a chemical reduction method associated with microwave irradiation to guarantee better controlled and uniform heating. The innovative Quality by Design strategy was implemented to optimise the different parameters of the process. A preliminary investigation design was firstly carried out to evaluate the influence of four parameters selected by means of an Ishikawa diagram. The critical quality attributes were to maximise the intensity of the SERS response and minimise its variance. The reaction time, temperature and stirring speed are critical process parameters. These were optimised using an I-optimal design. A robust operating zone covering the optimal reaction conditions (3.36 min-130 °C-600 rpm) associated with a probability of success was modelled. Validation of this point confirmed the prediction with intra- and inter-batch variabilities of less than 15%. In conclusion, this study successfully optimised silver nanoparticles by a rapid, low cost and simple technique enhancing the quantitative perspectives of SERS.

Quantitative Structure Retention-Relationship Modeling: Towards an Innovative General-Purpose Strategy.

Reversed-Phase Liquid Chromatography (RPLC) is a common liquid chromatographic mode used for the control of pharmaceutical compounds during their drug life cycle. Nevertheless, determining the optimal chromatographic conditions that enable this separation is time consuming and requires a lot of lab work. Quantitative Structure Retention Relationship models (QSRR) are helpful for doing this job with minimal time and cost expenditures by predicting retention times of known compounds without performing experiments. In the current work, several QSRR models were built and compared for their adequacy in predicting the retention times. The regression models were based on a combination of linear and non-linear algorithms such as Multiple Linear Regression, Support Vector Regression, Least Absolute Shrinkage and Selection Operator, Random Forest, and Gradient Boosted Regression. Models were built for five pH conditions, i.e., at pH 2.7, 3.5, 6.5, and 8.0. In the end, the model predictions were combined using stacking and the performances of all models were compared. The k-nearest neighbor-based application domain filter was established to assess the reliability of the prediction for further compound prioritization. Altogether, this study can be insightful for analytical chemists working with RPLC to begin with the computational prediction modeling such as QSRR to predict the separation of small molecules.

Interpretable One-Class Classification of Raman Spectra Using Prediction Bands Estimated by Wavelet Regression.

Previously, we introduced a novel one-class classification (OCC) concept for spectra. It uses as acceptance space for genuine spectra of the target chemical, a prediction band in the wavelengths' space. As a decision rule, test spectra falling substantially outside this band are rejected as noncomplying with the target, and their deviations are documented in the wavelengths' space. This band-based OCC concept was applied to smooth signals like near-infrared (NIR) spectra. A regression model based on a smoothed principal component (PC) representation of the training spectra was used to predict unseen trajectories of future spectra. The boundaries of the most central predicted trajectories were chosen as critical trajectories. We now propose a methodology to construct a similar band-based one-class classifier for Raman spectra, which are sharper and noisier than NIR spectra. The spectra are transformed by a composition of wavelet and principal component (wPC) expansions instead of just a PC expansion in the previous methodology for NIR spectra. Wavelets can capture sharp features of Raman signals and provide a framework to efficiently denoise them. A multinormal prediction model is then used to derive predictions of future wPC scores of unseen spectra. These predicted wPC scores are then backtransformed to obtain predictions of future trajectories of unseen spectra in the wavelengths' space, whose most central region defines the acceptance band or space. This band-based one-class classifier successfully classified the first derivatives of real pharmaceutical Raman spectra, while enjoying the advantage of documenting deviations from the critical trajectories in the wavelengths' space and hence is more interpretable.

A new alternative tool to analyse glycosylation in pharmaceutical proteins based on infrared spectroscopy combined with nonlinear support vector regression.

Almost 60% of commercialized pharmaceutical proteins are glycosylated. Glycosylation is considered a critical quality attribute, as it affects the stability, bioactivity and safety of proteins. Hence, the development of analytical methods to characterise the composition and structure of glycoproteins is crucial. Currently, existing methods are time-consuming, expensive, and require significant sample preparation steps, which can alter the robustness of the analyses. In this work, we suggest the use of a fast, direct, and simple Fourier transform infrared spectroscopy (FT-IR) combined with a chemometric strategy to address this challenge. In this context, a database of FT-IR spectra of glycoproteins was built, and the glycoproteins were characterised by reference methods (MALDI-TOF, LC-ESI-QTOF and LC-FLR-MS) to estimate the mass ratio between carbohydrates and proteins and determine the composition in monosaccharides. The FT-IR spectra were processed first by Partial Least Squares Regression (PLSR), one of the most used regression algorithms in spectroscopy and secondly by Support Vector Regression (SVR). SVR has emerged in recent years and is now considered a powerful alternative to PLSR, thanks to its ability to flexibly model nonlinear relationships. The results provide clear evidence of the efficiency of the combination of FT-IR spectroscopy, and SVR modelling to characterise glycosylation in therapeutic proteins. The SVR models showed better predictive performances than the PLSR models in terms of RMSECV, RMSEP, R2CV, R2Pred and RPD. This tool offers several potential applications, such as comparing the glycosylation of a biosimilar and the original molecule, monitoring batch-to-batch homogeneity, and in-process control.

Risk-taking bias in human decision-making is encoded via a right-left brain push-pull system.

A person's decisions vary even when options stay the same, like when a gambler changes bets despite constant odds of winning. Internal bias (e.g., emotion) contributes to this variability and is shaped by past outcomes, yet its neurobiology during decision-making is not well understood. To map neural circuits encoding bias, we administered a gambling task to 10 participants implanted with intracerebral depth electrodes in cortical and subcortical structures. We predicted the variability in betting behavior within and across patients by individual bias, which is estimated through a dynamical model of choice. Our analysis further revealed that high-frequency activity increased in the right hemisphere when participants were biased toward risky bets, while it increased in the left hemisphere when participants were biased away from risky bets. Our findings provide electrophysiological evidence that risk-taking bias is a lateralized push-pull neural system governing counterintuitive and highly variable decision-making in humans.

A New Alternative Tool to Analyse Glycosylation in Monoclonal Antibodies Based on Drop-Coating Deposition Raman imaging: A Proof of Concept.

Glycosylation is considered a critical quality attribute of therapeutic proteins as it affects their stability, bioactivity, and safety. Hence, the development of analytical methods able to characterize the composition and structure of glycoproteins is crucial. Existing methods are time consuming, expensive, and require significant sample preparation, which can alter the robustness of the analyses. In this context, we developed a fast, direct, and simple drop-coating deposition Raman imaging (DCDR) method combined with multivariate curve resolution alternating least square (MCR-ALS) to analyze glycosylation in monoclonal antibodies (mAbs). A database of hyperspectral Raman imaging data of glycoproteins was built, and the glycoproteins were characterized by LC-FLR-MS as a reference method to determine the composition in glycans and monosaccharides. The DCDR method was used and allowed the separation of excipient and protein by forming a "coffee ring". MCR-ALS analysis was performed to visualize the distribution of the compounds in the drop and to extract the pure spectral components. Further, the strategy of SVD-truncation was used to select the number of components to resolve by MCR-ALS. Raman spectra were processed by support vector regression (SVR). SVR models showed good predictive performance in terms of RMSECV, R2CV.

User-Driven Strategy for In Silico Screening of Reversed-Phase Liquid Chromatography Conditions for Known Pharmaceutical-Related Small Molecules.

In the pharmaceutical field, and more precisely in quality control laboratories, robust liquid chromatographic methods are needed to separate and analyze mixtures of compounds. The development of such chromatographic methods for new mixtures can result in a long and tedious process even while using the design of experiments methodology. However, developments could be accelerated with the help of in silico screening. In this work, the usefulness of a strategy combining response surface methodology (RSM) followed by multicriteria decision analysis (MCDA) applied to predictions from a quantitative structure-retention relationship (QSRR) model is demonstrated. The developed strategy shows that selecting equations for the retention time prediction models based on the pKa of the compound allows flexibility in the models. The MCDA developed is shown to help to make decisions on different criteria while being robust to the user's decision on the weights for each criterion. This strategy is proposed for the screening phase of the method lifecycle. The strategy offers the possibility to the user to select chromatographic conditions based on multiple criteria without being too sensitive to the importance given to them. The conditions with the highest desirability are defined as the starting point for further optimization steps.

Comparison of Vibrational Spectroscopic Techniques for Quantification of Water in Natural Deep Eutectic Solvents.

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.

Detection and Characterization of VIM-52, a New Variant of VIM-1 from a Klebsiella pneumoniae Clinical Isolate.

Over the last two decades, antimicrobial resistance has become a global health problem. In Gram-negative bacteria, metallo-ß-lactamases (MBLs), which inactivate virtually all ß-lactams, increasingly contribute to this phenomenon. The aim of this study is to characterize VIM-52, a His224Arg variant of VIM-1, identified in a Klebsiella pneumoniae clinical isolate. VIM-52 conferred lower MICs to cefepime and ceftazidime compared to VIM-1. These results were confirmed by steady-state kinetic measurements, where VIM-52 yielded a lower activity toward ceftazidime and cefepime but not against carbapenems. Residue 224 is part of the L10 loop (residues 221 to 241), which borders the active site. As Arg 224 and Ser 228 both play an important and interrelated role in enzymatic activity, stability, and substrate specificity for the MBLs, targeted mutagenesis at both positions was performed and further confirmed their crucial role for substrate specificity.

An exploratory data analysis method for identifying brain regions and frequencies of interest from large-scale neural recordings.

High-resolution whole brain recordings have the potential to uncover unknown functionality but also present the challenge of how to find such associations between brain and behavior when presented with a large number of regions and spectral frequencies. In this paper, we propose an exploratory data analysis method that sorts through a massive quantity of multivariate neural recordings to quickly extract a subset of brain regions and frequencies that encode behavior. This approach combines existing tools and exploits low-rank approximation of matrices without a priori selection of regions and frequency bands for analysis. In detail, the spectral content of neural activity across all frequencies of each recording contact is computed and represented as a matrix. Then, the rank-1 approximation of the matrix is computed using singular value decomposition and the associated singular vectors are extracted. The temporal singular vector, which captures the salient features of the spectrogram, is then correlated to the trial-varying behavioral signal. The distribution of correlations for each brain region is efficiently computed and used to find a subset of regions and frequency bands of interest for further examination. As an illustration, we apply this approach to a data set of local field potentials collected using stereoelectroencephalography from a human subject performing a reaching task. Using the proposed procedure, we produced a comprehensive set of brain regions and frequencies related to our specific behavior. We demonstrate how this tool can produce preliminary results that capture neural patterns related to behavior and aid in formulating data-driven hypotheses, hence reducing the time it takes for any scientist to transition from the exploratory to the confirmatory phase.

An intracerebral exploration of functional connectivity during word production.

Language is mediated by pathways connecting distant brain regions that have diverse functional roles. For word production, the network includes a ventral pathway, connecting temporal and inferior frontal regions, and a dorsal pathway, connecting parietal and frontal regions. Despite the importance of word production for scientific and clinical purposes, the functional connectivity underlying this task has received relatively limited attention, and mostly from techniques limited in either spatial or temporal resolution. Here, we exploited data obtained from depth intra-cerebral electrodes stereotactically implanted in eight epileptic patients. The signal was recorded directly from various structures of the neocortex with high spatial and temporal resolution. The neurophysiological activity elicited by a picture naming task was analyzed in the time-frequency domain (10-150 Hz), and functional connectivity between brain areas among ten regions of interest was examined. Task related-activities detected within a network of the regions of interest were consistent with findings in the literature, showing task-evoked desynchronization in the beta band and synchronization in the gamma band. Surprisingly, long-range functional connectivity was not particularly stronger in the beta than in the high-gamma band. The latter revealed meaningful sub-networks involving, notably, the temporal pole and the inferior frontal gyrus (ventral pathway), and parietal regions and inferior frontal gyrus (dorsal pathway). These findings are consistent with the hypothesized network, but were not detected in every patient. Further research will have to explore their robustness with larger samples.

Genetic and Biochemical Characterization of OXA-519, a Novel OXA-48-Like ß-Lactamase.

A multidrug-resistant Klebsiella pneumoniae 1210 isolate with reduced carbapenem susceptibility revealed the presence of a novel plasmid-encoded blaOXA-48-like gene, named blaOXA-519 The 60.7-kb plasmid (pOXA-519) was similar to the IncL-OXA-48 prototypical plasmid except for a ca. 2-kb deletion due to an IS1R insertion. OXA-519 differed from OXA-48 by a Val120Leu substitution, which resulted in an overall reduced ß-lactam-hydrolysis profile, except those for ertapenem and meropenem, which were increased. Thus, detection of OXA-519 producers using biochemical tests that monitor imipenem hydrolysis will be difficult.

Modeling the interactions between stimulation and physiologically induced APs in a mammalian nerve fiber: dependence on frequency and fiber diameter.

Electrical stimulation of nerve fibers is used as a therapeutic tool to treat neurophysiological disorders. Despite efforts to model the effects of stimulation, its underlying mechanisms remain unclear. Current mechanistic models quantify the effects that the electrical field produces near the fiber but do not capture interactions between action potentials (APs) initiated by stimulus and APs initiated by underlying physiological activity. In this study, we aim to quantify the effects of stimulation frequency and fiber diameter on AP interactions involving collisions and loss of excitability. We constructed a mechanistic model of a myelinated nerve fiber receiving two inputs: the underlying physiological activity at the terminal end of the fiber, and an external stimulus applied to the middle of the fiber. We define conduction reliability as the percentage of physiological APs that make it to the somatic end of the nerve fiber. At low input frequencies, conduction reliability is greater than 95% and decreases with increasing frequency due to an increase in AP interactions. Conduction reliability is less sensitive to fiber diameter and only decreases slightly with increasing fiber diameter. Finally, both the number and type of AP interactions significantly vary with both input frequencies and fiber diameter. Modeling the interactions between APs initiated by stimulus and APs initiated by underlying physiological activity in a nerve fiber opens opportunities towards understanding mechanisms of electrical stimulation therapies.

Poplar-Root Knot Nematode Interaction: A Model for Perennial Woody Species.

Plant root-knot nematode (RKN) interaction studies are performed on several host plant models. Though RKN interact with trees, no perennial woody model has been explored so far. Here, we show that poplar (Populus tremula × P. alba) grown in vitro is susceptible to Meloidogyne incognita, allowing this nematode to penetrate, to induce feeding sites, and to successfully complete its life cycle. Quantitative reverse transcription-polymerase chain reaction analysis was performed to study changes in poplar gene expression in galls compared with noninfected roots. Three genes (expansin A, histone 3.1, and asparagine synthase), selected as gall development marker genes, followed, during poplar-nematode interaction, a similar expression pattern to what was described for other plant hosts. Downregulation of four genes implicated in the monolignol biosynthesis pathway was evidenced in galls, suggesting a shift in the phenolic profile within galls developed on poplar roots. Raman microspectroscopy demonstrated that cell walls of giant cells were not lignified but mainly composed of pectin and cellulose. The data presented here suggest that RKN exercise conserved strategies to reproduce and to invade perennial plant species and that poplar is a suitable model host to study specific traits of tree-nematode interactions.

Uncertainty management for In Silico screening of reversed-phase liquid chromatography methods for small compounds.

The process of developing new reversed-phase liquid chromatography methods can be both time-consuming and challenging. To meet this challenge, statistics-based strategies have emerged as cost-effective, efficient and flexible solutions. In the present study, we use a Bayesian response surface methodology, which takes advantage of the knowledge of the pKa values of the compounds present in the analyzed sample to model their retention behavior. A multi-criteria decision analysis (MCDA) was then developed to exploit the uncertainty information inherent in the model distributions. This strategic approach is designed to integrate seamlessly with quantitative structure retention relationship (QSRR) models, forming an initial in-silico screening phase. Of the two methods presented for MCDA, one showed promising results. The method development process was carried out with the optimization phase, generating a design space that corroborates the results of the selection phase.

Development and optimization of a one step process for the production and sterilization of liposomes using supercritical CO2.

Liposomes are very interesting drug delivery systems for pharmaceutical and therapeutic purposes. However, liposome sterilization as well as their industrial manufacturing remain challenging. Supercritical carbon dioxide is an innovative technology that can potentially overcome these limitations. The aim of this study was to optimize a one-step process for producing and sterilizing liposomes using supercritical CO2. For this purpose, a design of experiment was conducted. The analysis of the experimental design showed that the temperature is the most influential parameter to achieve the sterility assurance level (SAL) required for liposomes (≤10-6). Optimal conditions (80 °C, 240 bar, 30 min) were identified to obtain the fixed critical quality attributes of liposomes. The conditions for preparing and sterilizing empty liposomes of various compositions, as well as liposomes containing the poorly water-soluble drug budesonide, were validated. The results indicate that the liposomes have appropriate physicochemical characteristics for drug delivery, with a size of 200 nm or less and a PdI of 0.35 or less. Additionally, all liposome formulations demonstrated the required SAL and sterility at concentrations of 5 and 45 mM, with high encapsulation efficiency.

Field assessment of active ingredient quantity in pharmaceutical tablets with limited calibration of near infrared spectra: An application to ciprofloxacin tablets.

Portable near-infrared (NIR) spectrophotometers have emerged as valuable tools for identifying substandard and falsified pharmaceuticals (SFPs). Integration of these devices with chemometric and machine learning models enhances their ability to provide quantitative chemical insights. However, different NIR spectrophotometer models vary in resolution, sensitivity, and responses to environmental factors such as temperature and humidity, necessitating instrument-specific libraries that hinder the wider adoption of NIR technology. This study addresses these challenges and seeks to establish a robust approach to promote the use of NIR technology in post-market pharmaceutical analysis. We developed support vector machine and partial least squares regression models based on binary mixtures of lab-made ciprofloxacin and microcrystalline cellulose, then applied the models to ciprofloxacin dosage forms that were assayed with high performance liquid chromatography (HPLC). A receiver operating characteristic (ROC) analysis was performed to set spectrophotometer independent NIR metrics to evaluate ciprofloxacin dosage forms as "meets standard," "needs HPLC assay," or "fails standard." Over 200 ciprofloxacin tablets representing 50 different brands were evaluated using spectra acquired from three types of NIR spectrophotometer with 85% of the prediction agreeing with HPLC testing. This study shows that non-brand-specific predictive models can be applied across multiple spectrophotometers for rapid screening of the conformity of pharmaceutical active ingredients to regulatory standard.

Nociceptor spontaneous activity is responsible for fragmenting non-rapid eye movement sleep in mouse models of neuropathic pain.

Spontaneous pain, a major complaint of patients with neuropathic pain, has eluded study because there is no reliable marker in either preclinical models or clinical studies. Here, we performed a comprehensive electroencephalogram/electromyogram analysis of sleep in several mouse models of chronic pain: neuropathic (spared nerve injury and chronic constriction injury), inflammatory (Freund's complete adjuvant and carrageenan, plantar incision) and chemical pain (capsaicin). We find that peripheral axonal injury drives fragmentation of sleep by increasing brief arousals from non-rapid eye movement sleep (NREMS) without changing total sleep amount. In contrast to neuropathic pain, inflammatory or chemical pain did not increase brief arousals. NREMS fragmentation was reduced by the analgesics gabapentin and carbamazepine, and it resolved when pain sensitivity returned to normal in a transient neuropathic pain model (sciatic nerve crush). Genetic silencing of peripheral sensory neurons or ablation of CGRP+ neurons in the parabrachial nucleus prevented sleep fragmentation, whereas pharmacological blockade of skin sensory fibers was ineffective, indicating that the neural activity driving the arousals originates ectopically in primary nociceptor neurons and is relayed through the lateral parabrachial nucleus. These findings identify NREMS fragmentation by brief arousals as an effective proxy to measure spontaneous neuropathic pain in mice.

A multi-target QSRR approach to model retention times of small molecules in RPLC.

Quantitative structure-retention relationship models (QSRR) have been utilized as an alternative to costly and time-consuming separation analyses and associated experiments for predicting retention time. However, achieving 100 % accuracy in retention prediction is unrealistic despite the existence of various tools and approaches. The limitations of vast data availability and time complexity hinder the use of most algorithms for retention prediction. Therefore, in this study, we examined and compared two approaches for modelling retention time using a dataset of small molecules with retention times obtained at multiple conditions, referred to as multi-targets (five pH levels: 2.7, 3.5, 5, 6.5, and 8 at gradient times of 20 min of mobile phase). The first approach involved developing separate models for predicting retention time at each condition (single-target approach), while the second approach aimed to learn a single model for predicting retention across all conditions simultaneously (multi-target approach). Our findings highlight the advantages of the multi-target approach over the single-target modelling approach. The multi-target models are more efficient in terms of size and learning speed compared to the single-target models. These retention prediction models offer two-fold benefits. Firstly, they enhance knowledge and understanding of retention times, identifying molecular descriptors that contribute to changes in retention behaviour under different pH conditions. Secondly, these approaches can be extended to address other multi-target property prediction problems, such as multi-quantitative structure Property(X) relationship studies (mt-QS(X)R).

A modelling study to dissect the potential role of voltage-gated ion channels in activity-dependent conduction velocity changes as identified in small fiber neuropathy patients.

Objective: Patients with small fiber neuropathy (SFN) suffer from neuropathic pain, which is still a therapeutic problem. Changed activation patterns of mechano-insensitive peripheral nerve fibers (CMi) could cause neuropathic pain. However, there is sparse knowledge about mechanisms leading to CMi dysfunction since it is difficult to dissect specific molecular mechanisms in humans. We used an in-silico model to elucidate molecular causes of CMi dysfunction as observed in single nerve fiber recordings (microneurography) of SFN patients. Approach: We analyzed microneurography data from 97 CMi-fibers from healthy individuals and 34 of SFN patients to identify activity-dependent changes in conduction velocity. Using the NEURON environment, we adapted a biophysical realistic preexisting CMi-fiber model with ion channels described by Hodgkin-Huxley dynamics for identifying molecular mechanisms leading to those changes. Via a grid search optimization, we assessed the interplay between different ion channels, Na-K-pump, and resting membrane potential. Main results: Changing a single ion channel conductance, Na-K-pump or membrane potential individually is not sufficient to reproduce in-silico CMi-fiber dysfunction of unchanged activity-dependent conduction velocity slowing and quicker normalization of conduction velocity after stimulation as observed in microneurography. We identified the best combination of mechanisms: increased conductance of potassium delayed-rectifier and decreased conductance of Na-K-pump and depolarized membrane potential. When the membrane potential is unchanged, opposite changes in Na-K-pump and ion channels generate the same effect. Significance: Our study suggests that not one single mechanism accounts for pain-relevant changes in CMi-fibers, but a combination of mechanisms. A depolarized membrane potential, as previously observed in patients with neuropathic pain, leads to changes in the contribution of ion channels and the Na-K-pump. Thus, when searching for targets for the treatment of neuropathic pain, combinations of several molecules in interplay with the membrane potential should be regarded.