Decolorization of textile azo dye Novacron Red using bacterial monoculture and consortium: Response surface methodology optimization.

The present study was intended toward the optimization of a textile dye Novacron Red decolorization by single and mixed culture of Bacillus strains namely, B. firmus, B. filamentosus and B. subterraneus. Optimization of dye decolorization using Bacillus monocultures was conducted using central composite design. The maximum dye decolorization achieved under optimized conditions for B. firmus, B. filamentosus and B. subterraneus was 89.24%, 88.28% and 88.45%, respectively. The effect of various consortia of selected Bacillus strains on dye removal was evaluated by applying a mixture design. The best dye (100 mg/L) decolorization yield (84%) was achieved using the consortium of B. filamentosus and B. subetrraneus.The Fourier Transform Infrared Spectroscopy analyses confirmed biodegradation potential of the two Bacillus strains. The results highlighted the potential of mono- and co-cultures of Bacillus strains for application in textile wastewater treatment. PRACTITIONER POINTS: Novel dye-decolorizing Bacillus strains were isolated from marine sediment. Optimization of decolorization was conducted using response surface methodology. Efficient decolorization of textile dye by Bacillus strains on mono- and co-cultures. The efficiency of the consortium B. filamentosus and B. subetrraneus on dye removal.

Neuropeptidomics: Comparison of parallel reaction monitoring and data-independent acquisition for the analysis of neuropeptides using high-resolution mass spectrometry.

Targeted peptide quantitation by mass spectrometry is a rapidly emerging field. Traditionally it relied on the development and validation of multiple reaction monitoring assays that could comply with a high level of sensitivity, specificity, accuracy and reproducibility in complex biological samples. However, with the introduction of high-resolution mass spectrometers, other acquisition modes could provide more comprehensive datasets for identification and quantification but also for in-depth data mining. The objective of this study was to evaluate two analytical approaches, parallel-reaction monitoring (PRM) and data-independent analysis (DIA) using a hybrid Quadrupole-Orbitrap mass spectrometer for the quantification of neuropeptides in animal spinal cord tissues. Mouse spinal cord tissues were harvested, homogenized and neuropeptides extracted using a C18 solid-phase extraction protocol. Chromatography was achieved using a Thermo Biobasic C8 100 × 1 mm (5 µm) column. The initial mobile phase conditions consisted of acetonitrile and water (both containing 0.1% of formic acid) at a ratio of 5:95. An 11 min linear gradient was applied up to a ratio of 50:50 and maintained for 3 min. The flow rate was fixed at 75 µL/min and 2 µL of sample was injected. Mass spectrometry analyses were performed using a Thermo Q Exactive Plus MS using PRM and DIA approaches. Quantitative data using an isotopic dilution and a label-free strategy were obtained for both methods and statistically compared. Using both approaches, we were able to clearly detect endogenous neuropeptides. However, with DIA, mass spectra alone could not distinguish Leu-Enk and Met-Enk. We used a Bland-Altman plot (Difference plot) to analyze the agreement between both approaches and no systematic bias was observed. Further statistical analyses, including variance analysis, showed more variability in DIA compared with PRM mode. Further analyses were performed using a label-free approach and confirmed an increase of the variance using a DIA approach.

Targeted high-resolution quadrupole-Orbitrap mass spectrometry analyses reveal a significant reduction of tachykinin and opioid neuropeptides level in PC1 and PC2 mutant mouse spinal cords.

Tachykinin and opioid neuropeptides play a fundamental role in pain transmission, modulation and inhibition. The proteolysis control of endogenous tachykinin and opioid neuropeptides has a significant impact on pain perception. The role of proprotein convertases (PCs) in the proteolysis of proneuropeptides was previously established but very few studies have shown the direct impact of PCs on the regulation of specific tachykinin and opioid peptides in the central nervous system. There is an increasing interest in the therapeutic targeting of PCs for the treatment of pain but it is imperative to assess the impact of PCs on the pronociceptive and the endogenous opioid systems. The objective of this study was to determine the relative concentration of targeted neuropeptides in the spinal cord of WT, PC1-/+ and PC2-/+ animals to establish the impact of a restricted PCs activity on the regulation of specific neuropeptides. The analysis of tachykinin and opioid neuropeptides were performed on a HPLC-MS/MS (High-Resolution Quadrupole-Orbitrap Mass Spectrometer). 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) concentrations in both, PC1-/+ and PC2-/+ animals. Therefore, the modulation of PCs activity has an important impact on specific pronociceptive peptides (SP and NKA), but the results also shown that endogenous opioid system is hindered and consequently it will affect significantly the pain modulatory pathways. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Characterization of Substance P processing in mouse spinal cord S9 fractions using high-resolution Quadrupole-Orbitrap mass spectrometry.

Tachykinins are a family of pronociceptive neuropeptides with a specific role in pain and inflammation. Several mechanisms regulate endogenous tachykinins and Substance P (SP) levels, including the differential expression of protachykinin mRNA and the controlled secretion of tachykinins from neurons. Proteolysis is suspected to regulate extracellular SP concentrations but few studies were conducted on the metabolism of proneuropeptides and neuropeptides. Here, we provide evidence that proteolysis controls SP levels in the spinal cord leading to the formation of active C-terminal fragments. Using high-resolution mass spectrometry, specific tachykinins fragments were characterized and quantified. The metabolic stability of ß-Tachykinin58-71 and SP were very short resulting in half-life of 5.7 and 3.5min respectively. Several C-terminal fragments were identified, including SP3-11, SP5-11 and SP8-11, which conserve affinity for the Neurokinin 1 receptor. Interestingly, the metabolic stability of C-terminal fragments was 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.

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 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 min 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 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.

Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1(-/-) mouse spinal cords reveals 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 an 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 affects 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.