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Background: Micro-RNAs could provide great insights about the neuropathological mechanisms associated with osteoarthritis (OA) pain processing. Using the validated Montreal Induction of Rat Arthritis Testing (MI-RAT) model, this study aimed to characterize neuroepigenetic markers susceptible to correlate with innovative pain functional phenotype and targeted neuropeptide alterations. Methods: Functional biomechanical, somatosensory sensitization (peripheral-via tactile paw withdrawal threshold; central-via response to mechanical temporal summation), and diffuse noxious inhibitory control (via conditioned pain modulation) alterations were assessed sequentially in OA (n = 12) and Naïve (n = 12) rats. Joint structural, targeted spinal neuropeptides and differential expression of spinal cord micro-RNAs analyses were conducted at the sacrifice (day (D) 56). Results: The MI-RAT model caused important structural damages (reaching 35.77% of cartilage surface) compared to the Naïve group (P < 0.001). This was concomitantly associated with nociceptive sensitization: ipsilateral weight shift to the contralateral hind limb (asymmetry index) from -55.61% ± 8.50% (D7) to -26.29% ± 8.50% (D35) (P < 0.0001); mechanical pain hypersensitivity was present as soon as D7 and persisting until D56 (P < 0.008); central sensitization was evident at D21 (P = 0.038); pain endogenous inhibitory control was distinguished with higher conditioned pain modulation rate (P < 0.05) at D7, D21, and D35 as a reflect of filtrated pain perception. Somatosensory profile alterations of OA rats were translated in a persistent elevation of pro-nociceptive neuropeptides substance P and bradykinin, along with an increased expression of spinal miR-181b (P = 0.029) at D56. Conclusion: The MI-RAT OA model is associated, not only with structural lesions and static weight-bearing alterations, but also with a somatosensory profile that encompasses pain centralized sensitization, associated to active endogenous inhibitory/facilitatory controls, and corresponding neuropeptidomic and neuroepigenetic alterations. This preliminary neuroepigenetic research confirms the crucial role of pain endogenous inhibitory control in the development of OA chronic pain (not only hypersensitivity) and validates the MI-RAT model for its study.
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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 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 neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. 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, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.
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The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.
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Antiprotozoários , Antiprotozoários/farmacologia , Antiprotozoários/química , Esfingolipídeos/metabolismo , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Hexosiltransferases/antagonistas & inibidores , Leishmania/efeitos dos fármacos , Leishmania/genética , Leishmania/enzimologia , Animais , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/genética , Leishmania mexicana/enzimologia , Glicoesfingolipídeos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Understanding the endocannabinoid system in C. elegans may offer insights into basic biological processes and potential therapeutic targets for managing pain and inflammation in human. It is well established that anandamide modulates pain perception by binding to cannabinoid and vanilloid receptors, regulating neurotransmitter release and neuronal activity. One objective of this study was to demonstrate the suitability of C. elegans as a model organism for assessing the antinociceptive properties of bioactive compounds and learning about the role of endocannabinoid system in C. elegans. The evaluation of the compound anandamide (AEA) revealed antinociceptive activity by impeding C. elegans nocifensive response to noxious heat. Proteomic and bioinformatic investigations uncovered several pathways activated by AEA. Enrichment analysis unveiled significant involvement of ion homeostasis pathways, which are crucial for maintaining neuronal function and synaptic transmission, suggesting AEA's impact on neurotransmitter release and synaptic plasticity. Additionally, pathways related to translation, protein synthesis, and mTORC1 signaling were enriched, highlighting potential mechanisms underlying AEA's antinociceptive effects. Thermal proteome profiling identified NPR-32 and NPR-19 as primary targets of AEA, along with OCR-2, Cathepsin B, Progranulin, Transthyretin, and ribosomal proteins. These findings suggest a complex interplay between AEA and various cellular processes implicated in nociceptive pathways and inflammation modulation. Further investigation into these interactions could provide valuable insights into the therapeutic potential of AEA and its targets for the management of pain-related conditions.
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Ácidos Araquidônicos , Caenorhabditis elegans , Endocanabinoides , Alcamidas Poli-Insaturadas , Canais de Cátion TRPV , Animais , Caenorhabditis elegans/metabolismo , Endocanabinoides/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Alcamidas Poli-Insaturadas/farmacologia , Canais de Cátion TRPV/metabolismo , Ácidos Araquidônicos/metabolismo , Ácidos Araquidônicos/farmacologia , Receptores de Canabinoides/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Temperatura Alta , Analgésicos/farmacologiaRESUMO
The development of interventions targeting reservoirs of Borrelia burgdorferi sensu stricto with acaricide to reduce the density of infected ticks faces numerous challenges imposed by ecological and operational limits. In this study, the pharmacokinetics, efficacy and toxicology of fluralaner were investigated in Mus musculus and Peromyscus leucopus mice, the main reservoir of B. burgdorferi in North America. Fluralaner showed rapid distribution and elimination, leading to fast plasma concentration (Cp) depletion in the first hours after administration followed by a slow elimination rate for several weeks, resulting in a long terminal half-life. Efficacy fell below 100% while Cp (± standard deviation) decreased from 196 ± 54 to 119 ± 62 ng/mL. These experimental results were then used in simulations of fluralaner treatment for a duration equivalent to the active period of Ixodes scapularis larvae and nymphs. Simulations showed that doses as low as 10 mg/kg have the potential to protect P. leucopus against infestation for a full I. scapularis active season if administered at least once every 7 days. This study shows that investigating the pharmacology of candidate acaricides in combination with pharmacokinetic simulations can provide important information to support the development of effective interventions targeting ecological reservoirs of Lyme disease. It therefore represents a critical step that may help surpass limits inherent to the development of these interventions.
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Acaricidas , Borrelia burgdorferi , Reservatórios de Doenças , Ixodes , Doença de Lyme , Peromyscus , Animais , Doença de Lyme/tratamento farmacológico , Camundongos , Ixodes/microbiologia , Ixodes/efeitos dos fármacos , Reservatórios de Doenças/microbiologia , Peromyscus/microbiologia , Acaricidas/farmacocinética , Acaricidas/farmacologia , Borrelia burgdorferi/efeitos dos fármacos , Isoxazóis/farmacocinética , FemininoRESUMO
BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.
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Antiprotozoários , Leishmania infantum , Leishmaniose Visceral , Humanos , Animais , Cães , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Antimônio/farmacologia , Antimônio/uso terapêutico , Espécies Reativas de Oxigênio , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/veterinária , Resistência a MedicamentosRESUMO
Cannflavins, flavonoids abundantly present in Cannabis sativa, possess a distinct chemical structure comprising a vanillyl group. Notably, the capsaicin structure also contains a vanillyl group, which is considered essential for interacting with the vanilloid receptor. The vanilloid receptor plays a crucial role in the perception of pain, heat, and inflammation and mediates the analgesic effects of capsaicin. Therefore, we postulated that prolonged exposure to cannflavin A (Can A) and cannflavin B (Can B) would provoke vanilloid receptor desensitization and hinder nocifensive responses to noxious thermal stimuli. C. elegans wild-type (N2) and mutants were exposed to Can A and Can B solutions for 60 min and then aliquoted on Petri dishes divided into quadrants for thermal stimulation. We then determined the thermal avoidance index for each C. elegans experimental group. Proteomics was performed to identify proteins and pathways associated with Can A or B treatment. Prolonged exposure to Can A and Can B hindered heat avoidance (32-35 °C) in C. elegans. No antinociceptive effect was observed 6 h post Can A or B exposure. Proteomics and Reactome pathway enrichment analyses identified hierarchical differences between Can A- and B-treated nematodes. However, both treatments were related to eukaryotic translation initiation (R-CEL-72613) and metabolic processes strongly associated with pain development. Our study aids in characterizing the pharmacological activity of cannflavins isolated from Cannabis sativa and outlines a possible application as pain therapy.
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Caenorhabditis elegans , Cannabis , Animais , Capsaicina , Temperatura Alta , Canais de Cátion TRPV , DorRESUMO
Cannabis has gained popularity in recent years as a substitute treatment for pain following the risks of typical treatments uncovered by the opioid crisis. The active ingredients frequently associated with pain-relieving effects are the phytocannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), but their effectiveness and mechanisms of action are still under research. In this study, we used Caenorhabditis elegans, an ideal model organism for the study of nociception that expresses mammal ortholog cannabinoid (NPR-19 and NPR-32) and vanilloid (OSM-9 and OCR-2) receptors. Here, we evaluated the antinociceptive activity of THC and CBD, identifying receptor targets and several metabolic pathways activated following exposure to these molecules. The thermal avoidance index was used to phenotype each tested C. elegans experimental group. The data revealed for the first time that THC and CBD decreases the nocifensive response of C. elegans to noxious heat (32-35 °C). The effect was reversed 6 h post- CBD exposure but not for THC. Further investigations using specific mutants revealed CBD and THC are targeting different systems, namely the vanilloid and cannabinoid systems, respectively. Proteomic analysis revealed differences following Reactome pathways and gene ontology biological process database enrichment analyses between CBD or THC-treated nematodes and provided insights into potential targets for future drug development.
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Proteínas de Caenorhabditis elegans , Canabidiol , Canabinoides , Humanos , Animais , Canabidiol/farmacologia , Dronabinol/farmacologia , Caenorhabditis elegans , Proteômica , Dor , Analgésicos/farmacologia , Mamíferos , Receptores Acoplados a Proteínas GRESUMO
Validating animal pain models is crucial to enhancing translational research and response to pharmacological treatment. This study investigated the effects of a calibrated slight exercise protocol alone or combined with multimodal analgesia on sensory sensitivity, neuroproteomics, and joint structural components in the MI-RAT model. Joint instability was induced surgically on day (D) 0 in female rats (N = 48) distributed into sedentary-placebo, exercise-placebo, sedentary-positive analgesic (PA), and exercise-PA groups. Daily analgesic treatment (D3-D56) included pregabalin and carprofen. Quantitative sensory testing was achieved temporally (D-1, D7, D21, D56), while cartilage alteration (modified Mankin's score (mMs)) and targeted spinal pain neuropeptide were quantified upon sacrifice. Compared with the sedentary-placebo (presenting allodynia from D7), the exercise-placebo group showed an increase in sensitivity threshold (p < 0.04 on D7, D21, and D56). PA treatment was efficient on D56 (p = 0.001) and presented a synergic anti-allodynic effect with exercise from D21 to D56 (p < 0.0001). Histological assessment demonstrated a detrimental influence of exercise (mMs = 33.3%) compared with sedentary counterparts (mMs = 12.0%; p < 0.001), with more mature transformations. Spinal neuropeptide concentration was correlated with sensory sensitization and modulation sites (inflammation and endogenous inhibitory control) of the forced mobility effect. The surgical MI-RAT OA model coupled with calibrated slight exercise demonstrated face and predictive validity, an assurance of higher clinical translatability.
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Neuropeptídeos , Osteoartrite , Animais , Feminino , Roedores , Dor/tratamento farmacológico , Osteoartrite/patologia , Neuropeptídeos/uso terapêutico , Analgésicos/farmacologiaRESUMO
The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.
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Neoplasias da Mama , Doenças do Cão , Neoplasias Pulmonares , Glândulas Mamárias Humanas , Neoplasias Mamárias Animais , Humanos , Animais , Cães , Camundongos , Feminino , Fluvastatina/uso terapêutico , Fatores de Transcrição/metabolismo , Xenoenxertos , Glândulas Mamárias Humanas/metabolismo , Linhagem Celular Tumoral , Doenças do Cão/tratamento farmacológico , Doenças do Cão/metabolismo , Neoplasias da Mama/veterinária , Neoplasias Mamárias Animais/tratamento farmacológico , Neoplasias Mamárias Animais/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/veterinária , Necrose/veterinária , Doxorrubicina , RNA MensageiroRESUMO
Staphylococcus aureus is one of the major pathogens causing bovine mastitis, and antibiotic treatment is most often inefficient due to its virulence and antibiotic-resistance attributes. The development of new antibiotics for veterinary use should account for the One Health concept, in which humans, animals, and environmental wellbeing are all interconnected. S. aureus can infect cattle and humans alike and antibiotic resistance can impact both if the same classes of antibiotics are used. New effective antibiotic classes against S. aureus are thus needed in dairy farms. We previously described PC1 as a novel antibiotic, which binds the S. aureus guanine riboswitch and interrupts transcription of essential GMP synthesis genes. However, chemical instability of PC1 hindered its development, evaluation, and commercialization. Novel PC1 analogs with improved stability have now been rationally designed and synthesized, and their in vitro and in vivo activities have been evaluated. One of these novel compounds, PC206, remains stable in solution and demonstrates specific narrow-spectrum activity against S. aureus. It is active against biofilm-embedded S. aureus, its cytotoxicity profile is adequate, and in vivo tests in mice and cows show that it is effective and well tolerated. PC206 and structural analogs represent a promising new antibiotic class to treat S. aureus-induced bovine mastitis.
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LY-404,039 is an orthosteric agonist of metabotropic glutamate 2 and 3 receptors (mGluR2/3) that may harbour additional agonist effect at dopamine D2 receptors. LY-404,039 and its pro-drug, LY-2140023, have previously entered clinical trials as treatment options for schizophrenia. They could therefore be repurposed, if proven efficacious, for other conditions, notably Parkinson's disease (PD). We have previously shown that the mGluR2/3 orthosteric agonist LY-354,740 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. Unlike LY-404,039, LY-354,740 does not stimulate dopamine D2 receptors, suggesting that LY-404,039 may elicit broader therapeutic effects in PD. Here, we sought to investigate the effect of this possible additional dopamine D2-agonist action of LY-404,039 by assessing its efficacy on dyskinesia, PLBs and parkinsonism in the MPTP-lesioned marmoset. We first determined the pharmacokinetic profile of LY-404,039 in the marmoset, in order to select doses resulting in plasma concentrations known to be well tolerated in the clinic. Marmosets were then injected L-DOPA with either vehicle or LY-404,039 (0.1, 0.3, 1 and 10 mg/kg). The addition of LY-404,039 10 mg/kg to L-DOPA resulted in a significant reduction of global dyskinesia (by 55%, P < 0.01) and PLBs (by 50%, P < 0.05), as well as reduction of global parkinsonism (by 47%, P < 0.05). Our results provide additional support of the efficacy of mGluR2/3 orthosteric stimulation at alleviating dyskinesia, PLBs and parkinsonism. Because LY-404,039 has already been tested in clinical trials, it could be repurposed for indications related to PD.
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Discinesia Induzida por Medicamentos , Doença de Parkinson , Transtornos Parkinsonianos , Transtornos Psicóticos , Animais , Levodopa/farmacologia , Antiparkinsonianos/farmacologia , Antiparkinsonianos/uso terapêutico , Callithrix , Dopamina , Comportamento Animal , Discinesia Induzida por Medicamentos/tratamento farmacológico , Discinesia Induzida por Medicamentos/etiologia , Transtornos Parkinsonianos/tratamento farmacológico , Transtornos Parkinsonianos/induzido quimicamente , Doença de Parkinson/tratamento farmacológico , Transtornos Psicóticos/tratamento farmacológico , 1-Metil-4-Fenil-1,2,3,6-Tetra-HidropiridinaRESUMO
Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was previously shown as differentially expressed in granulosa cells (GC) of bovine pre-ovulatory follicles suggesting that JAK3 could modulate GC function and activation/inhibition of downstream targets. We used JANEX-1, a JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3, CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GC were treated with or without FSH in the presence or not of JANEX-1. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited. Proliferation markers CCND2 and PCNA were reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses revealed phosphorylation of specific amino acid residues within JAK3 as well as CDKN1B and MAPK8IP3 suggesting possible activation or inhibition post-FSH or JANEX-1 treatments. We show that FSH activates JAK3 in GC, which could phosphorylate target proteins and likely modulate other signaling pathways involving CDKN1B and MAPK8IP3, therefore controlling GC proliferation and steroidogenic activity.
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Hormônio Foliculoestimulante , Janus Quinases , Animais , Bovinos , Feminino , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/metabolismo , Janus Quinase 3/metabolismo , Janus Quinases/metabolismo , Fosforilação , Transdução de Sinais , Fatores de Transcrição STAT/metabolismo , Espectrometria de Massas em TandemRESUMO
Vanilloids, including capsaicin and eugenol, are ligands of transient receptor potential channel vanilloid subfamily member 1 (TRPV1). Prolonged treatment with vanilloids triggered the desensitization of TRPV1, leading to analgesic or antinociceptive effects. Caenorhabditis elegans (C. elegans) is a model organism expressing vanilloid receptor orthologs (e.g., OSM-9 and OCR-2) that are associated with behavioral and physiological processes, including sensory transduction. We have shown that capsaicin and eugenol hamper the nocifensive response to noxious heat in C. elegans. The objective of this study was to perform proteomics to identify proteins and pathways responsible for the induced phenotype and to identify capsaicin and eugenol targets using a thermal proteome profiling (TPP) strategy. The results indicated hierarchical differences following Reactome Pathway enrichment analyses between capsaicin- and eugenol-treated nematodes. However, both treated groups were associated mainly with signal transduction pathways, energy generation, biosynthesis and structural processes. Wnt signaling, a specific signal transduction pathway, is involved following treatment with both molecules. Wnt signaling pathway is noticeably associated with pain. The TPP results show that capsaicin and eugenol target OCR-2 but not OSM-9. Further protein-protein interaction (PPI) analyses showed other targets associated with enzymatic catalysis and calcium ion binding activity. The resulting data help to better understand the broad-spectrum pharmacological activity of vanilloids.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Capsaicina/farmacologia , Eugenol/farmacologia , Transdução de Sinais , Canais de Cátion TRPV/metabolismo , Analgésicos/química , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
Bitopertin, a selective glycine transporter 1 (GlyT1) inhibitor, has been extensively studied for the treatment of schizophrenia, with known safety and tolerability profiles in the clinic. Whereas several rodent experiments have been reported, the pharmacokinetic (PK) profile of bitopertin in rodents has not been extensively reported, as only two studies disclosed limited PK parameters in male rats after oral administration. Here, we determined the PK profile of bitopertin in female Sprague-Dawley rats. Blood samples were taken serially, before and after sub-cutaneous (0.03, 0.1, 0.3, 1, and 3 mg/kg) or intra-venous (0.1 mg/kg) administration. Plasma levels were determined by high-performance liquid chromatography coupled with heat-assisted electrospray ionisation tandem mass spectrometry (HPLC-HESI MS/MS). Subsequently, PK parameters were calculated using non-compartmental analysis, including area under the curve (AUC), time (Tmax) to maximal plasma concentration (Cmax), clearance (CL), volume of distribution (Vz), as well as half-life (T1/2). Following sub-cutaneous injection, bitopertin exhibited dose-dependent AUC0-∞ (439.6-34,018.9 ng/mL) and Tmax (3.7-24.0 h), a very long terminal T1/2 (35.06-110.32 h) and low CL (0.07-0.13 L/h/kg), suggesting that bitopertin is slowly absorbed and eliminated in the rat. The observed relationship between dose and the extent of drug exposure (AUC) was linear. Following administration of all sub-cutaneous doses, measured bitopertin plasma levels were comparable to levels achieved with doses already administered in the clinic. We hope that our results will be useful in the design of pre-clinical experiments in which this drug will eventually be administered sub-cutaneously.
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Piperazinas , Espectrometria de Massas em Tandem , Masculino , Ratos , Feminino , Animais , Ratos Sprague-Dawley , Sulfonas , Administração OralRESUMO
ABSTRACT: Neuropathic pain is a complex, debilitating disease that results from injury to the somatosensory nervous system. The presence of systemic chronic inflammation has been observed in patients with chronic pain but whether it plays a causative role remains unclear. This study aims to determine the perturbation of systemic homeostasis by an injury to peripheral nerve and its involvement in neuropathic pain. We assessed the proteomic profile in the serum of mice at 1 day and 1 month after partial sciatic nerve injury (PSNL) or sham surgery. We also assessed mouse mechanical and cold sensitivity in naïve mice after receiving intravenous administration of serum from PSNL or sham mice. Mass spectrometry-based proteomic analysis revealed that PSNL resulted in a long-lasting alteration of serum proteome, where most of the differentially expressed proteins were in inflammation-related pathways, involving cytokines and chemokines, autoantibodies, and complement factors. Although transferring sham serum to naïve mice did not change their pain sensitivity, PSNL serum significantly lowered mechanical thresholds and induced cold hypersensitivity in naïve mice. With broad anti-inflammatory properties, bone marrow cell extracts not only partially restored serum proteomic homeostasis but also significantly ameliorated PSNL-induced mechanical allodynia, and serum from bone marrow cell extracts-treated PSNL mice no longer induced hypersensitivity in naïve mice. These findings clearly demonstrate that nerve injury has a long-lasting impact on systemic homeostasis, and nerve injury-associated systemic inflammation contributes to the development of neuropathic pain.
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Neuralgia , Proteômica , Camundongos , Animais , Nervo Isquiático/lesões , Neuralgia/etiologia , Hiperalgesia/metabolismo , Inflamação/metabolismoRESUMO
Targeted mass spectrometry is extensively used for the quantitative measurement of various molecules present in complex matrices. It is certainly one of the most important analytical duties in a mass spectrometry laboratory. Systematic development of selected-reaction monitoring (SRM), multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) methods for targeted mass spectrometry-based analysis was performed without considering future opportunities. The advancement of hardware and software technologies has resulted in greater resolution, accuracy, speed and depth. For sure, SRM, MRM or PRM acquisitions can quantify molecules very accurately at trace levels. However, they do not provide datasets allowing future data mining. Obviously, we cannot truly quantify something that we do not know is there. However, using non-targeted data acquisition for target analysis, we can generate a MS1 and MS2 digital libraries of each sample, providing future proof datasets. This is instrumental for data mining following new questions potentially arising in time permitting new and deeper processing and interpretation. This perspective article provides thoughts on why we believe it is time to question the status quo in targeted mass spectrometry.
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Proteômica , Proteômica/métodos , Espectrometria de Massas/métodosRESUMO
Extracellular vesicles (EVs) contribute to intercellular communication through the transfer of their rich cargo to recipient cells. The EVs produced by LPS-stimulated neutrophils from healthy humans and horses increase airway smooth muscle (ASM) proliferation, but the roles of neutrophil EVs in asthma are largely unexplored. The aim of this study was to determine whether neutrophil-derived EVs isolated during the remission or exacerbation of asthma influence ASM proliferation differentially. Peripheral blood neutrophils were collected during remission and exacerbation in eight horses affected by severe asthma. The cells were cultured (±LPS), and their EVs were isolated by ultracentrifugation and characterized by laser scattering microscopy and proteomic analysis. The proliferation of ASM co-incubated with EVs was monitored in real time by electrical impedance. Two proteins were significantly upregulated during disease exacerbation in neutrophil EVs (MAST4 and Lrch4), while LPS stimulation greatly altered the proteomic profile. Those changes involved the upregulation of neutrophil degranulation products, including proteases known to induce myocyte proliferation. In agreement with the proteomic results, EVs from LPS-stimulated neutrophils increased ASM proliferation, without an effect of the disease status. The inhalation of environmental LPS could contribute to asthma pathogenesis by activating neutrophils and leading to ASM hyperplasia.
Assuntos
Asma , Vesículas Extracelulares , Humanos , Cavalos , Animais , Neutrófilos/metabolismo , Proteômica , Lipopolissacarídeos/farmacologia , Proliferação de Células , Músculo Liso/metabolismo , Asma/patologia , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos , Proteínas Serina-Treonina QuinasesRESUMO
Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.
Assuntos
Antiprotozoários , Leishmania infantum , Anfotericina B/farmacologia , Antimônio/metabolismo , Antimônio/farmacologia , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Proteoma/análise , ProteômicaRESUMO
RATIONALE: The COVID-19 pandemic demonstrated the importance of high-throughput analysis for public health. Given the importance of surface viral proteins for interactions with healthy tissue, they are targets of interest for mass spectrometry-based analysis. For that reason, the possibility of detecting and quantifying peptides using a high-throughput technique, laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS), was explored. METHODS: Two peptides used as models for small peptides (leu-enkephalin and endomorphin-2) and four tryptic peptides (GVYYPDK, NIDGYFK, IADYNYK, and QIAPGQTGK) specific to the SARS-CoV-2 Spike protein were employed. Target peptides were analyzed individually in the positive mode by LDTD-QqQMS. Peptides were quantified by internal calibration using selected reaction monitoring transitions in pure solvents and in samples spiked with 20 µg mL-1 of a bovine serum albumin tryptic digest to represent real analysis conditions. RESULTS: Low-energy fragment ions (b and y ions) as well as high-energy fragment ions (c and x ions) and some of their corresponding water or ammonia losses were detected in the full mass spectra. Only for the smallest peptides, leu-enkephalin and endomorphin-2, were [M + H]+ ions observed. Product ion spectra confirmed that, with the experimental conditions used in the present study, LDTD transfers a considerable amount of energy to the target peptides. Quantitative analysis showed that it was possible to quantify peptides using LDTD-QqQMS with acceptable calibration curve linearity (R2 > 0.99), precision (RSD < 18.2%), and trueness (bias < 8.3%). CONCLUSIONS: This study demonstrated for the first time that linear peptides can be qualitatively and quantitatively analyzed using LDTD-QqQMS. Limits of quantification and dynamic ranges are still inadequate for clinical applications, but other applications where higher levels of proteins must be detected could be possible with LDTD. Given the high-throughput capabilities of LDTD-QqQMS (>15 000 samples in less than 43 h), more studies are needed to improve the sensitivity for peptide analysis of this technique.