Liquid Chromatography-Electrospray Linear Ion Trap Mass Spectrometry Analysis of Targeted Neuropeptides in Tac1-/- Mouse Spinal Cords Reveal Significant Lower Concentration of Opioid Peptides

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop a HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1-/- mouse endogenous opioid system is hampered and therefore affect significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1-/- spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p < 0.0001). No significant concentration differences were observed in mouse Tac1-/- spinal cords for Met-Enk and CGRP. The analysis of Tac1-/- mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies

Étude de la régulation des tachykinines et son impact sur l’expression des peptides opioïdes à l'aide de la chromatographie liquide à haute performance et de la spectrométrie de masse

Les peptides appartenant à la famille des tachykinines telle que la substance P (SP) sont des acteurs essentiels contribuant à l’hyperalgésie primaire et secondaire. La SP libérée par les neurones afférents primaires, ne provoque pas à elle seule des décharges nociceptives, mais elle potentialise l’effet de divers neurotransmetteurs tel que le glutamate. Pour ces différentes raisons, de nombreuses recherches ont été effectuées avec des antagonistes des récepteurs neurokinines et en particulier des récepteurs NK1. Cependant, malgré des études pré-cliniques prometteuses, les antagonistes du récepteur NK1 n’ont pas montré d’effet significatif chez l’Homme. La biosynthèse des neuropeptides actifs passe par la maturation protéolytique des pro-neuropeptides. La compréhension des mécanismes de la maturation enzymatique des précurseurs des tachykinines, ainsi que l’étude de la stabilité métabolique de la substance P (SP) permettraient d’élucider des stratégies de traitement innovateur en favorisant l’inhibition du processus de maturation ou la production de fragments peptidiques moins actifs ou inactifs. Le premier objectif de cette étude était d’élucider le rôle de la Proproteine convertase 1 (PC1) et de la Proproteine convertase 2 (PC2) dans la maturation de la protachykinine en utilisant des fractions S9 de la moelle épinière des souris du type sauvage (WT), PC1-/+ et PC2-/+. La caractérisation et la quantification des neuropeptides ont été réalisées à l’aide de la chromatographie liquide à haute performance et de la spectrométrie de masse. Les résultats montrent que PC1 et PC2 interviennent dans la maturation de la protachykinine et ces deux enzymes sont essentielles pour la biosynthèse de la Tachykinine58-71, le précurseur de la SP. Une réduction de plus de 50% de la vitesse de formation dans les fractions S9 de la moelle épinière de souris mutantes PC1 et PC2 a été observée. Les résultats obtenus révèlent que PC1 et PC2 sont impliquées dans la protéolyse de la protachykinine et suggèrent un rôle important de ces enzymes dans la maturation de la protachykinine-1. La protéolyse régule probablement les concentrations extracellulaires de la SP, mais peu d'études ont été menées sur le métabolisme des tachykinines. Dans ce présent travail, nous démontrons que la protéolyse contrôle le niveau de la SP dans la moelle épinière menant à la formation de fragments C-terminaux actifs. La stabilité métabolique de la β-tachykinine58-71 et de la SP était très courte, avec une demi-vie de 5.7 et 3.5 min, respectivement. Plusieurs fragments C-terminaux ont été identifiés, y compris la SP3-11, la SP5-11 et la SP8-11, qui conservent leurs affinités vis-à-vis des récepteurs neurokinines. La stabilité métabolique des fragments C-terminaux était significativement supérieure à celle de la β-Tachykinine58-71 et de la SP. Deux inhibiteurs de Prolyl endopeptidase spécifiques ont été utilisés et ont montré une réduction significative de la vitesse de formation de SP3-11 et de SP5-11. Ainsi, nous avons démontré que le Prolyl endopeptidase est impliqué dans le traitement N-terminal de la SP dans la moelle épinière et dans la formation de la SP3-11 et la SP5-11. Étant donné que la régulation des niveaux endogènes de peptides opioïdes (DynA, Leu-Enk, Met-Enk) et des tachykinines (Tach58-71, SP) dépend fondamentalement de l'activité de PC1 et de celle de PC2, l'analyse des tachykinines et des neuropeptides opioïdes ont été réalisées. Les résultats obtenus révèlent une diminution significative des neuropeptides pro nociceptifs la Tach58-71 (p <0,05), de la SP (p <0,01) et du NKA (P <0,001)), et des neuropeptides opioïdes DynA (p <0,01), de Leu-Enk (p <0,001), de Met-Enk (p <0,001), dans la moelle épinière de souris PC1 - / + et PC2 - / +. Par conséquent, la modulation de l'activité des PCs a un impact important sur les peptides pro-nociceptifs, mais également sur le système opioïde endogène et par conséquent elle affectera significativement les voies modulatrices de la douleur. Ces résultats suggèrent également que la réduction significative des concentrations de peptides pro-nociceptifs peut altérer la réponse du système opioïde endogène. Les analyses des concentrations des peptides opioïdes chez les souris Tac1-/- ont montré spécifiquement que les concentrations en Endomorphine-2 (EM2), en Leu-Enk et en Dyn A sont significativement inférieures que celles obtenues dans la moelle épinière chez les souris WT. Par conséquent, l’absence de la SP a un impact sur les mécanismes endogènes de modulation de la douleur. Mots clés : Tachykinines, substance P, proprotéines convertases, protéolyse, peptides opioïdes, moelle épinière, douleur, chromatographie liquide à haute performance, spectrométrie de masse.SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons and plays a central role in nociceptive transmission. The SP does not acte alone to cause nociceptive discharges, but it potentiates the effect of various neurotransmitters such as glutamate. For these various reasons, much research has been carried out with antagonists of neurokinin receptors and in particular NK1 receptors. However, despite promising pre-clinical studies, NK1 receptor antagonists have not shown significant effect in Humans. The proteolysis control of endogenous protachykinins has a profound impact on pain perception. Proprotein convertases (PCs) are extensively expressed in the central nervous system and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue but the role of PCs remains unclear. The first objective of this study was to decipher the role of PC1 and PC2 in the proteolysis of protachykinins using cellular fractions of spinal cords from wild type (WT), PC1 -/+ and PC2 -/+ mices and mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and β-Tachykinin58-71, an important SP precursor, with over 50 % reduction of the rate of formation in mutant PC1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein. The proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. In the present study, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. The metabolic stability of β-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5 min, respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the neurokinin receptors. Interestingly, the metabolic stability of C-terminal fragments were significantly superior. Two specific Prolyl endopeptidase inhibitors were used and showed a significant reduction in the rate of formation of SP3-11 and SP5-11 providing strong evidence that Prolyl endopeptidase is involved into N-terminal processing of SP in the spinal cord. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. This study has determined the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1- / + and PC2- /+ mice to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The results revealed a significant decrease of Dyn A (p < 0.01), Leu-Enk (p < 0.001), Met-Enk (p < 0.001), Tach58-71 (p < 0.05), SP (p < 0.01) and NKA (p < 0.001) spinal cord concentrations in both, PC1 -/+ and PC2 -/+ mice. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also showed that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1-/- spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. Our results reveal that Endomorphin-2 (EM2), Leu-Enk and Dyn A were down regulated in Tac1-/- spinal cord tissues that strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These results may have insightful impact on future analgesic drug developments and therapeutic strategies. Key words: Tachykinins, substance P, proprotein convertases, proteolysis, opioid peptides, spinal cord, pain, high performance liquid chromatography, mass spectrometry

Tachykinins Processing is Significantly Impaired in PC1 and PC2 Mutant Mouse Spinal Cord S9 Fractions

Substance P (SP) play a central role in nociceptive transmission and it is an agonist of the Neurokinin-1 receptor located in the lamina I of the spinal cord. SP is a major proteolytic product of the protachykinin-1 primarily synthesized in neurons. Proprotein convertases (PCs) are extensively expressed in the central nervous system (CNS) and specifically cleave at C-terminal of either a pair of basic amino acids, or a single basic residue. The proteolysis control of endogenous protachykinins has a profound impact on pain perception and the role of PCs remain unclear. The objective of this study was to decipher the role of PC1 and PC2 in the proteolysis surrogate protachykinins (i.e. Tachykinin 20-68 and Tachykinin 58-78) using cellular fractions of spinal cords from wild type (WT), PC1-/+ and PC2-/+ animals and mass spectrometry. Full-length Tachykinin 20-68 and Tachykinin 58-78 was incubated for 30 minutes in WT, PC1-/+ and PC2-/+ mouse spinal cord S9 fractions and specific C-terminal peptide fragments were identified and quantified by mass spectrometry. The results clearly demonstrate that both PC1 and PC2 mediate the formation of SP and Tachykinin 58-71, an important SP precursor, with over 50% reduction of the rate of formation in mutant PC 1 and PC2 mouse S9 spinal cord fractions. The results obtained revealed that PC1 and PC2 are involved in the C-terminal processing of protachykinin peptides and suggest a major role in the maturation of the protachykinin-1 protein

Dermatomyosite du sujet âgé: étude de 4 observations dans le sud tunisien

La dermatomyosite (DM) touche essentiellement l’adolescent et l’adulte jeune, elle est très rare chez le sujet âgé, le plus souvent associée à des complications iatrogènes et à une pathologie cancéreuse. Nous avons étudié les caractéristiques de la DM du sujet âgé à travers une étude rétrospective dans laquelle nous avons comparé 4 patients âgés de plus de 65 ans au début de la myosite avec 40 sujets jeunes.Pan African Medical Journal 2012; 13:2

Les tumeurs primitives de la glande submandibulaire: à propos de 25 cas

Les tumeurs des glandes salivaires sont rares, elles constituent 3% des tumeurs de la tête et du cou et 0,6% des tumeurs humaines. La pathologie tumorale de la glande sous-maxillaire est moins fréquente que celle de la parotide et pose autant de problèmes aussi bien diagnostiques que thérapeutiques, surtout l'opportunité de la radiothérapie post-opératoire et l'attitude vis-à-vis des récidives. L'objectif de notre étude est de discuter les problèmes diagnostiques, les résultats thérapeutiques et évolutifs que présentent ces tumeurs afin d'en dégager l'attitude thérapeutique la plus appropriée en se basant sur une étude rétrospectif incluant une série de 25 cas de tumeurs de la glande sous maxillaire colligées aux services d'orl et au service de radiothérapie du CHU MED VI de Marrakech sur une période allant de Janvier 2009 jusqu'au décembre 2014. L'étude a concerné 09 hommes et 18 femmes. La moyenne d'âge était de 48 ans (25 - 71 ans), La tuméfaction sous mandibulaire était le principal symptôme révélateur. Le caractère douloureux a été signalé par 09 patients. Une seule paralysie du rameau mentonnier du nerf facial a été objectivée au moment de l'examen chez un malade. Les adénopathies cervicales ont été retrouvées chez 09 patients. La localisation jugulocarotidienne haute homolatérale est la plus fréquente. Sur le plan thérapeutique, une sous maxillectomie a été réalisée chez 24 malades dont 02 réopéré pour récidive d'un adénome pléomorphe et un patient pour un carcinome adénoïde kystique. 3 tumeurs malignes n'ont pu bénéficier d'aucun traitement chirurgical du fait du caractère explosif de la lésion Les résultats histopathologiques ont montré une répartition largement dominée par l'adénome pléomorphe pour les tumeurs bénignes (66,66% des tumeurs bénignes), et le carcinome adénoïde kystique pour les tumeurs malignes (37,5% des tumeurs malignes). Neuf malades de notre série ont été irradiés en post opératoire. Deux tumeurs bénignes ayant présenté une première récidive sont toutes des adénomes pléomorphes, 04 tumeurs malignes ont présenté une première récidive (03 carcinome adénoïde kystique, 01 carcinome epidermoide) et un carcinome adénoïde kystique a présenté une deuxième récidive après un complément radiothérapeutique. La pathologie tumorale de la glande sous-maxillaire est complexe, dominée par les tumeurs malignes, elle pose des problèmes diagnostiques et thérapeutiques; Son diagnostic est orienté par des arguments cliniques et radiologiques, repose sur l'analyse anatomopathologique de la pièce d'exérèse opératoire. Un retard diagnostic joint a un traitement initial inadéquat assombrit d'avantage son pronostic.Pan African Medical Journal 2014; 22

Synergistic Interaction of Rhizobium tropici, Rhizophagus irregularis and Serendipita indica in Promoting Snap Bean Growth

The overuse of chemical pesticides and fertilizers in crop farming has led to a decrease in crop quality and negative impacts on soil and the environment. It is crucial to adopt alternative strategies to maintain soil and environmental quality while enhancing crop growth and yield. To explore this, a study was conducted under greenhouse conditions to investigate the effect of Rhizobium tropici CIAT 899 alone, as well as in association with mycorrhizae (Rhizophagus irregularis) and endophytic fungus (Serendipita indica), on the growth, yield, and nutrient status of snap bean plants. At harvest, the rhizobial strain CIAT 899 demonstrated the highest effectiveness. It significantly increased the number of nodules in both Contender and Garrafal Enana varieties by 6.97% and 14.81%, respectively, compared with the control without inoculation. Furthermore, the results indicated that co-inoculation of Rhizobium and symbiotic fungi had positive effects on nitrogen content, phosphorus availability, and overall plant growth. Regardless of the variety, plants inoculated with R. tropici CIAT 899 and Serendipita indica exhibited the highest values for plant growth parameters. This combination resulted in 168% and 135% increases in root dry biomass, as well as 140% and 225% increases in the number of pods for Contender and Garrafal Enana, respectively, compared with the control at harvest. Additionally, this study highlights the potential benefits of combining R. tropici with either Serendipita indica or Rhizophagus irregularis in terms of nitrogen and phosphorus uptake. These symbiotic microorganisms demonstrated synergistic interactions with snap bean plants, leading to improved mineral nutrition and enhanced growth. Overall, these findings suggest that utilizing these symbiotic microorganisms can effectively enhance the mineral nutrition and growth of snap bean plants.info:eu-repo/semantics/publishedVersio

Testing the co-integration relationship between auto insurance premiums and risk compensation amount

This study investigates the influential economic factors impacting auto insurance premiums and explores the potential for a long-term equilibrium relationship. Monthly data from 25 branches of the National Insurance Company in the regional agency of Ouargla, Algeria, is analyzed using descriptive and case study methods. Through a thorough trend analysis and statistical model formulation, including co-integration tests, stability tests, and regression analyses, we assess the relationship between variables and the impact of independent variables on the dependent variable. The results reveal a statistically acceptable yet weak short-term connection, lacking evidence of common integration. This research provides insights into the complex dynamics of insurance premiums and compensation relationships, suggesting nuanced connections and guiding recommendations for adaptable premium structures and responsive strategies

What do you think of an unusual axillary mass?

Skin apocrine carcinoma is a rare malgnancy of epidermal adnexa, most frequent in axillary seat, where apocrine sweat gland are abundant, the neoplasm can arise in groin, anogenital, lips, eyelid, characterized by a plate or surface area of nodules hummocky. Etiology and incidence are not known. The prognosis is influenced by the risk of locoregional recurrence and metastatic evolution. We describe the case of 61-year-old man who presented a left axillary slow-growing mass since 2 years ago. The cutaneous biopsy objectified an apocrine adenocarcinoma. The paraclinic exams performed to detect primary breast were tumor negative, first step before confirming the diagnosis. Standard treatment is surgical excision with margins of 2 to 3cm for local tumor, for apocrine adenocarcinoma regional lymph node dissection if nodes were clinically positive is wide surgical excision. This kind of tumour is chemoresistant. In this case, adjuvant chemotherapy was indicated, before surgery to reduce tumoral volume. This case illustrates the importance clinicopathological correlation of skin cancer, particularly apocrine one. Clinical particularity and careful analyses histology helps diagnosis approach

Extraction and Characterization of Tunisian Quercus ilex Starch and Its Effect on Fermented Dairy Product Quality

In this study, a new starch has been isolated from acorn (Quercus ilex) fruits. The chemical composition of acorn flour showed its richness in carbohydrates (64.43%), proteins (8%), and fat (10%). The extraction yield of acorn starch was about 34.5%. Thus, the composition of extracted acorn starch and its physical and functional properties were studied. Acorn starch had high purity represented by low proportions of proteins (0.92%) and lipids (0.51%) with a pH of 5.3. The swelling power was 20.76 g/g, while the solubility was about 64.22% at 90°C which suggests that acorn starch has potential for use in food industries. The FT-IR spectra of isolated native starches have shown the main bands characterizing the starch. However, X-ray diffractograms exhibited an A- and B-type diffraction pattern. Furthermore, the effect of acorn starch incorporation at different levels (0.5%, 1%, 1.5%, and 2%) on the quality parameters of a fermented dairy product was investigated at the beginning of storage. The results demonstrated that the most suitable dose of acorn starch to be incorporated in the fermented dairy product was lower than 1%. This low concentration reduced syneresis, improved functional properties, and enhanced the viscosity of the fermented dairy product

Pemphigoid gestationis: Report of 44 cases

Introduction: Pemphigoid gestationis (PG) is a rare autoimmune bullous dermatosis occurring during pregnancy and post partum. The aim of our study was to analyse epidemio-clinical, immunopathological, therapeutic and prognostic features of PG through a hospital study of 44 cases. Methods: It is a retrospective study, collecting all cases diagnosed at the dermatological department of Farhat Hached Soussa (Tunisia) from January 1990 to December 2015. Results: The disease began in 40,9% of cases at the second trimester of pregnancy and 48% at the third trimester of pregnancy, one case in post abortum. It was a polymorphic eruption, localized in the chest, the back and limbs in all cases. The face was affected in 18,2% of cases and the mucosa in one case. The skin biopsy showed a subepidermal blister in 25 cases. The direct immunofluoresence found linear C3 deposits along the basement membrane in all cases, associated to IgG deposits in 25% of cases. The treatment was mainly based on local or general corticotherapy with favourable evolution. We noted exacerbation in post partum in 17 cases and relapses during the ulterior pregnancies in 7 cases. Fetal damage was present in PG in 8 cases. Conclusion: Our study is conformed to literature data; nevertheless, it is distinguished by its appearance on the face the less frequency of palmo-plantar manifestations and the exceptional case of PG postabortum