Abstract Gabapentin (GBP) and pregabalin (PGB) are widely used gabapentinoids. Previously, we have demonstrated, for the first time, that GBP and PGB modulate the nociceptive response to noxious heat in C. elegans at an optimal concentration. In the current study, we use C. elegans and paired thermal nociception assays with direct internal drug concentration measurements to characterize the pharmacokinetic (PK)/pharmacodynamic (PD) relationship of both compounds. Neither drug altered baseline mobility or quadrant preference, confirming that behavioral effects reflected genuine antinociceptive action. Both GBP and PGB produced dose- and time-dependent reductions in thermal avoidance, with 500 µM exposures generating a biphasic, V-shaped time course in which suppression of thermal sensitivity deepened before partially reversing. This partial reversal occurred later with PGB than with GBP. Internal concentrations confirmed dose-dependent absorption and retention for both drugs, yet at 500 µM, internal drug levels remained elevated through 360 min even as behavioral avoidance recovered, indicating that the recovery limb reflects active counter-regulation rather than passive clearance, consistent with previously reported transcriptional and proteomic signatures. Exposure–response profiles were notably flat, suggesting a saturable pharmacodynamic ceiling. Molecular modeling revealed conserved electronic pharmacophores supporting shared α2δ engagement, alongside shape-descriptor differences that may contribute to divergent absorption kinetics. These findings position C. elegans as a valuable model for dissecting gabapentinoid PK/PD relationships. Beyond mechanistic insight, these findings support the continued investigation of C. elegans as a screening platform whose validation could help address the 3R (Replacement, Reduction, Refinement) principles guiding animal research.

Pain modulation relies on complex molecular interactions among ion channels, G protein-coupled receptors, and intracellular signaling cascades. The Transient Receptor Potential Vanilloid 1 (TRPV1) channel serves as a polymodal detector and integrator of noxious stimuli, linking sensory transduction with broader neuromodulatory systems. This review delineates the mechanistic crosstalk between TRPV1, endocannabinoid, and opioid pathways in nociceptive regulation. TRPV1 activation by heat, protons, or endogenous lipids induces calcium influx and engages protein kinase C (PKC), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) pathways that modulate channel phosphorylation and neuronal excitability. Endocannabinoids such as anandamide act as dual CB1 and TRPV1 agonists, establishing feedback loops that adjust nociceptive thresholds, while μ-opioid receptor activation inhibits adenylate cyclase and TRPV1 sensitization through Gi/o-mediated signaling. Given the recent progress in cryo-electron microscopy and molecular modeling, simulation studies have been possible, revealing key structural determinants underlying key receptor interactions. Integrating pharmacophore modeling, molecular docking, and artificial intelligence-based screening enables rational design of multi-target ligands that exploit TRPV1-endocannabinoid-opioid synergy. This mechanistic framework supports the development of next-generation analgesics that achieve potent, sustained, and safe modulation of nociceptive signaling.

Chronic pain is a global burden, driving urgent demand for novel, effective, and non-addictive therapeutic strategies. The vanilloid receptor TRPV1 is a key nociceptive mediator in mammals, yet structural understanding of related channels in invertebrates remains unclear, limiting translational research efforts. Caenorhabditis elegans relies on TRPV-like proteins (OSM-9 and OCR-2) for chemical and thermal nociception, offering an opportunity to probe the conservation of interaction principles governing channel receptors across species. Using a combination of structural modelling, flexible docking, and molecular dynamics, we mapped capsaicin binding across homo- and heterotetrameric assemblies of TRPV1, OSM-9, and OCR-2. All receptors established capsaicin-binding complexes within conserved inter-subunit architectures. However, OSM-9 and OCR-2 exhibited distinct interaction chemistries, shifting away from the TRPV1-dominating polar and electrostatic contacts toward enriched aromatic, hydrogen bond, and sulfur-mediated contacts. Importantly, the OCR-2 binding site displayed the most favourable interaction profile, including conserved hydrogen-bonding networks at positions analogous to TRPV1's validated ligand-contact residues. These results reveal that TRPV-like channels preserve common structural features for vanilloid recognition even if different interaction chemistries drive ligand engagement. Our findings support OCR-2 binding as the primary capsaicin response in C. elegans and uncover architectural determinants that signal novel, unexplored regulatory elements in pain mechanisms.

Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that, in mice, changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell type-specific profiling revealed that spinal inhibitory and excitatory neurons exhibited substantial changes in translation after peripheral nerve injury. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity. Conversely, reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability but did not alleviate mechanical hypersensitivity. Together, these findings advance our understanding of translational control mechanisms in the spinal cord during neuropathic pain and highlight their cell type- and phase-specific contributions to gene expression and pain hypersensitivity.

Terpenes such as Limonene (LIMO) and β-Caryophyllene (BC) have demonstrated pain-modulating properties, potentially through interactions with the Transient receptor potential vanilloid 1 (TRPV1) receptor. This study examines the antinociceptive effects of four terpenes derived from Cannabis sativa: Limonene, β-Caryophyllene, α-Humulene (HUM), and α-Myrcene (MYR) using Caenorhabditis elegans (C. elegans). The primary objective was to characterize terpene-induced modulation of nocifensive responses to noxious heat, and to elucidate their influence on molecular pathways via specific receptor targets. Thermotaxis assays quantified the antinociceptive activity of increasing terpene concentrations in wild-type nematodes. To assess receptor-specific mechanisms, assays were performed in mutant strains lacking functional OCR-2 and OSM-9 (TRPV-like nociceptors) and NPR-19 and NPR-32 (encoding cannabinoid-like receptors). Proteomic profiling coupled with bioinformatics analysis identified terpene-induced alterations in signaling pathways and biological processes. All four terpenes exhibited significant antinociceptive activity in wild-type C. elegans, with impaired effects observed in vanilloid receptor mutants, implicating TRPV-like channels in their mechanism of action. Proteomic and pathway analyses revealed terpene-specific molecular signatures, highlighting differential modulation of neuronal and stress-responsive signaling cascades. By elucidating the molecular mechanisms underlying terpene-induced nociceptive modulation, this work strengthens the growing body of evidence supporting the therapeutic promise of terpenes in pain management outside the effect referred to as the "entourage effect".

ABSTRACT Although oxytetracycline (OTC) has been widely used in poultry since 1950, comprehensive studies characterizing its degradation products for potential therapeutic applications have not been conducted. The aims of the present study were to evaluate the antibacterial activity of three OTC degradation products (4-α-epi-OTC [4-EOTC], α-apo-OTC, and β-apo-OTC) individually and in combination with OTC, to identify and assess the genetic determinants conferring antimicrobial resistance (AMR) in avian pathogenic Escherichia coli (APEC) strains exposed to tetracyclines and OTC degradation products, and to assess OTC degradation in a simulated poultry gizzard. The microdilution method was used to determine the minimal inhibitory concentration (MIC) of OTC, chlortetracycline, tetracycline, 4-EOTC, α-apo-OTC, and β-apo-OTC against 15 APEC strains. The fractional inhibitory concentration index (FICI) was calculated to assess the combined effect of OTC with its degradation products. A conjugation assay was conducted to determine whether resistance to OTC and its active degradation products is mediated by the identified tetracycline resistance genes. Results showed that while α-apo-OTC and β-apo-OTC lacked detectable antibacterial activity, 4-EOTC retained inhibitory activity against all tested APEC strains. Notably, a synergistic effect was observed between OTC and 4-EOTC (FICI ≤ 0.5). Preliminary evidence indicated that E. coli employed the same resistance determinant ( tetA ) against both OTC and its degradation products, while confirming that OTC remained stable under simulated gizzard conditions. The identification of antibacterial activity in 4-EOTC represents a key finding of this study and warrants further investigation to define its activity spectrum and explore its potential therapeutic applications. IMPORTANCE Tetracyclines are among the most extensively used antimicrobials in veterinary medicine, with oxytetracycline (OTC) serving as a cornerstone compound of this family. While several studies have reported that OTC undergoes degradation in the environment, the biological roles of the resulting chemical derivatives in poultry remain poorly understood. The present study demonstrates that one major degradation product, 4-EOTC, retains antibacterial activity against avian pathogenic Escherichia coli (APEC) strains and, when combined with OTC, produces a synergistic effect. Importantly, the tetA gene confers resistance in APEC against 4-EOTC as it does against OTC, highlighting the possible contribution of OTC degradation products to the spread of antimicrobial resistance (AMR). Furthermore, OTC was found to remain stable under simulated poultry gizzard conditions, indicating that its degradation likely occurs in downstream compartments of the poultry gastrointestinal tract. Collectively, these findings reveal that OTC degradation products are not biologically inert and open new avenues for the development of next-generation tetracyclines.

Bacterial biofilms are structured communities of bacterial cells enclosed in a self-produced polymeric matrix, which can adhere to biotic or abiotic surfaces. This mode of existence permits these bacteria to endure in adverse conditions, including the presence of antibiotics Bacteria within biofilms are responsible for numerous infections in humans and animals, including bovine mastitis. It is therefore important to develop new therapeutic strategies to control and treat biofilm-associated infections. The results obtained by our group during the study of mixed bacterial biofilm communities showed that four isolates of coagulase-negative staphylococci (CNS; two Staphylococcus chromogenes and two Staphylococcus simulans ) that produce only a small amount of biofilm can significantly reduce biofilm formation in approximatively 80% of pathogenic staphylococci associated with bovine mastitis. Furthermore, supernatants of S. chromogenes reduced secondary intramammary colonization by S. aureus in a murine model of mastitis. However, information regarding the mechanism and the effector molecule(s) involved is lacking. The objective of this study was therefore to investigate and characterize the antibiofilm molecule(s) produced by these four CNS isolates. In this context, we prepared culture supernatants from two isolates of S. chromogenes (C and E) and two isolates of S. simulans (F and H) to evaluate their effect on biofilm production of pathogenic bacterial species involved in bovine mastitis. Using a standard biofilm microtiter plate assay, we demonstrated that the four CNS supernatants not only have a significant impact on biofilms of pathogenic staphylococci (68% of tested isolates) but also on those of other important mastitis pathogens such as Streptococcus spp., Trueperella pyogenes , Klebsiella spp. and Escherichia coli (61.3% of tested isolates) . The isolation and characterization of the antibiofilm molecule(s) contained in the supernatants were then conducted using a filtration process with membranes of different porosities, as well as through physicochemical and enzymatic treatments. We were then able to confirm that antibiofilm activity against staphylococci was present in the < 3kDa fractions of CNS culture supernatants and that this activity was heat-stable and protease-resistant, but sensitive to RNase A, suggesting that the antibiofilm activity might be due, at least in part, to an RNA molecule. Preliminary results showed that the antibiofilm activity was maintained with RNA extracts from fractioned supernatants (<3kDa). In conclusion, these results confirmed that some CNS have an antibiofilm activity, which represents a promising new avenue in the fight against biofilm-associated infections, particularly bovine mastitis.

Objective:To describe the pharmacokinetic parameters of tramadol and its main metabolites, O-desmethyltramadol (M1) and N-desmethyltramadol, and clinically detectable adverse effects after a single orally administered high dose of tramadol in rabbits (Oryctolagus cuniculus). Methods:6 experimental and 1 control healthy intact male rabbits of commercial origin were included in February 2025. Following administration of a 30-mg/kg oral dose of tramadol, plasma concentrations of tramadol, M1, and N-desmethyltramadol were determined by UHPLC-MS at 12 predetermined time points. Pharmacokinetic parameters were calculated using commercial software. Fecal production and sedation were evaluated before and after the experiment. Results:The mean tramadol maximum plasmatic concentration was 91 ± 38 ng/mL, the average time to reach maximum plasmatic concentration was 40 minutes, the terminal half-life was 4.0 ± 2.4 hours, and the mean area under the curve from the first dose to infinity was 192 ± 45 ng/hmL. The M1 metabolite reached concentrations compatible with previously described analgesic effects in rabbits after 10 minutes and for up to 3 hours after administration in some individuals, whereas tramadol did not reach analgesic concentrations. Mild sedation was detected in 4 rabbits at the 20 minute- to 6-hour time points, and fecal production increased from 24 to 48 hours after tramadol administration. No clinically relevant adverse effects were noted. Conclusions:Administration of 30 mg/kg tramadol, PO, in rabbits results in plasma concentrations of M1 compatible with analgesia. Clinical Relevance:The short duration of action warrants further studies with long-acting formulations of tramadol.

As individuals age, they often experience persistent, unresolved pain, impacting their quality of life. Aging as a process is accompanied by "inflammaging," a state of chronic, low-grade systemic inflammation contributing to various diseases. Understanding the functional link between inflammaging and age-related development of pain is crucial for identifying novel therapeutic targets. We hypothesized that the circulatory milieu plays a role in regulating pain and that inflammaging contributes to changes in pain behavior with age. To test these hypotheses, we monitored nociception and postsurgical pain in male and female mice aged 3 and 24 months and analyzed their serum proteome, including cytokine/chemokine profiles. Our results demonstrated that compared with young mice, aging mice were hyposensitive to mechanical stimulation, yet their pain response to incision was aggravated and prolonged. Serum proteomic analysis revealed sex-specific inflammaging patterns. To explore the link between inflammaging and age-related alteration in pain behavior, we applied a rejuvenation strategy by transferring serum from 3-month-old mice to 19- to 21-month-old mice. Young serum normalized mechanical sensitivity in aged mice, alleviated postsurgical mechanical pain, and promoted recovery. Alongside the improvements in pain behavior phenotype, young serum recalibrated the aging serum profile. It reduced age-associated increases of cytokine/chemokine levels in male mice and rescued age-related, female-selective downregulation of inflammatory pathways such as liver X receptor/retinoid X receptor activation, D24-dehydrocholesterol reductase, and complement signaling. Our findings suggest that the circulatory environment, notably inflammaging, plays a significant role in altered pain behavior of aging mice. The sex-specific signature of age-dependent systemic inflammation highlights the importance of investigating inflammaging through the lens of sexual dimorphism.

Neglected tropical diseases caused by trypanosomatid parasites present a major public healthcare issue, partly due to emerging resistance. Attachment of ω-alkynyl chains characteristic of the lipid tails of antiparasitic peptides to the p-position of anisomycin gave ethers exhibiting potent activity, rivalling that of the parent ribosomal inhibitor, especially against resistant Leishmania strains. Single-particle cryoelectron microscopy analysis revealed that O-propargyl anisomycin binds to the highly conserved peptidyl transferase center of the ribosome similar to the parent inhibitor. Thermal proteomic profiling and gene ontology analysis demonstrated that O-propargyl anisomycin exhibited a broader mode of action, including activity against glycosome-associated proteins. Alkynyl substituents improved antiparasitic activity against resistant strains, likely by enlarging the mode of action, offering a novel path toward therapy against trypanosomatid infections.

Terpenes such as Limonene and β-Caryophyllene have demonstrated pain-modulating properties, potentially through interactions with TRPV1 receptors. This study examines the antinociceptive effects of four terpenes derived from Cannabis sativa : Limonene, β-Caryophyllene, α-Humulene, and α-Myrcene using Caenorhabditis elegans ( C. elegans ). The primary objective was to characterize terpene-induced modulation of nocifensive responses to noxious heat, and to elucidate their influence on molecular pathways via specific receptor targets. Thermotaxis assays quantified the antinociceptive activity of increasing terpene concentrations in wild-type nematodes. To assess receptor-specific mechanisms, assays were performed in mutant strains lacking functional OCR-2 and OSM-9 (TRPV-like vanilloid nociceptors), and NPR-19 and NPR-32 (encoding cannabinoid-like receptors). Proteomic profiling coupled with bioinformatics analysis identified terpene-induced alterations in signaling pathways and biological processes. All four terpenes exhibited significant antinociceptive activity in wild-type C. elegans , with impaired effects observed in vanilloid receptor mutants, implicating TRPV-like channels in their mechanism of action. Proteomic and pathway analyses revealed terpene-specific molecular signatures, highlighting differential modulation of neuronal and stress-responsive signaling cascades. By elucidating the molecular mechanisms underlying terpene-induced nociceptive modulation, this work strengthens the growing body of evidence supporting the therapeutic promise of terpenes in pain management outside the effect referred to as the “entourage effect.”

Chronic pain remains a major unmet medical challenge, and lipid signaling pathways have emerged as key modulators of nociception. Using Caenorhabditis elegans as a genetically tractable model, we investigated how fatty acid composition influences thermal avoidance behavior. Mutant strains lacking functional desaturase enzymes ( elo-1, fat-1, fat-2, fat-3, fat-4, fat-6 / fat-7 ), and consequently depleted in polyunsaturated fatty acids (PUFAs) such as arachidonic acid, displayed significantly reduced sensitivity to noxious heat compared to wild-type animals. These findings indicate that intact PUFA biosynthesis is essential for normal thermal nociception in C. elegans . Given that arachidonic acid is a precursor of endocannabinoids (AEA and 2-AG) known to modulate TRPV1-dependent pain signaling, our results suggest that a conserved lipid-based mechanism regulates heat avoidance in nematodes. This study establishes a functional link between fatty acid metabolism and nociceptive behavior, providing a powerful platform to explore metabolic modulation of pain pathways. ### Competing Interest Statement The authors have declared no competing interest. National Sciences and Engineering Research Council of Canada, RGPIN-2020-05228 Canada Research Chair, CRC-2021-00160

Mass spectrometry is a critical tool to understand complex changes in biological processes. Despite significant advances in search engine technology, many spectra remain unassigned. This research evaluates the performance of three rescoring platforms, Oktoberfest, MS2Rescore, and inSPIRE, using MaxQuant output. The results indicated a substantial increase in identifications at the peptide level (40%-53%) and PSM level (64%-67%). However, some peptides were lost due to limitations in processing posttranslational modifications (PTMs)-with up to 75% of lost peptides exhibiting PTMs. Each platform displayed distinct strengths and weaknesses. For instance, inSPIRE performed best in terms of peptide identifications and unique peptides, while MS2Rescore performed better for PSMs at higher FDR values. Differences in platform performance stemmed from different sources: original search engine feature selection, type of ion series predicted, retention time predictor, and PTMs compatibility. Overall, inSPIRE showed a superior ability to harness original search engine results. Taken all together, rescoring platforms clearly outperformed original search results; however, they demanded additional computation time (up to 77%) and manual adjustments. The findings here underline the necessity of integrating rescoring platforms into current proteomics pipelines but also address some challenges in their implementation and optimization. Future integrated platforms may help enhance adoption.

AZD8529 is a highly selective metabotropic glutamate 2 (mGlu2) receptor positive allosteric modulator (PAM) that has undergone clinical trials for schizophrenia and smoking cessation. Previously, we demonstrated that the selective mGlu2 receptor PAMs LY-487,379, CBiPES, and biphenylindanone A (BINA) alleviated L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia and psychosis-like behaviours (PLBs) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset model of Parkinson's disease (PD). However, these drugs are not clinical candidates because of their pharmacological properties, contrary to AZD8529 which could be repurposed if pre-clinically efficacious. To assess the effect of AZD8529 on L-DOPA-induced dyskinesia and PLBs in the MPTP-lesioned marmoset, we first determined the pharmacokinetic (PK) profile of AZD8529 in this species to inform dose selection such that drug plasma levels were clinically relevant. Then, MPTP-lesioned animals were treated with L-DOPA with either vehicle or AZD8529 (0.1, 0.3, 1, and 10 mg/kg). The results showed a reduction in global dyskinesia severity (up to 70 %, P < 0.001), and in duration of on-time with disabling dyskinesia (up to 97 %, P < 0.001) when compared to L-DOPA/vehicle. Similarly, there was a reduction in global PLB severity (up to 64 %, P < 0.001), and in duration of on-time with disabling PLBs (up to 94 %, P < 0.001) when compared to L-DOPA/vehicle. Additionally, AZD8529 increased the duration of the anti-parkinsonian action of L-DOPA at doses of 0.3 mg/kg and above (up to 29 %, P < 0.05). Our results further demonstrate the potential of AZD8529 and mGlu2 receptor positive allosteric modulation for alleviating L-DOPA-induced dyskinesia and PLBs while amplifying the therapeutic efficacy of L-DOPA.

Rationale: Selection of proteomic workflows for a given project can be a daunting task. This research provides a guide outlining the impact on protein identification of different steps such as chromatographic separation, data acquisition strategies, and bioinformatic pipelines. The data presented here will help experts and nonexpert proteomic users to increase proteome coverage and peptide identification. Methods: HeLa protein digests were analyzed through different C18 chromatographic columns (15 and 50 cm in length), using top 12 data-dependent acquisition (DDA), top 20 DDA, and data-independent acquisition (DIA) with a nanospray source in positive mode in a Thermo Q Exactive instrument. The raw data were analyzed using different search engines, rescoring approaches, and multi-engine searches. The results were analyzed in the context of peptide and protein identifications, precursor properties, and computation requirements to understand the differences between methods. Results: Our results showed that higher column lengths and top N DDA approaches were able to significantly increase protein identifications. The use of multiple search engines yielded limited gains, whereas the use of rescoring methods clearly outperformed other strategies. Finally, DIA approaches, although successful at generating new identifications, had a limited performance influenced by the previous collection of DDA data, which could prohibitively increase instrument time. Nonetheless, the use of library-free methods showed promising results. Conclusions: Our results highlight the impact of different experimental approaches on proteome coverage. Changes in chromatographic columns, data acquisition, or bioinformatic analysis can significantly increase the number of protein identifications (>400%). Thus, this research provides a reference upon which to build a successful proteomic workflow with different considerations at every step.