Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

Research of Pesticide Metabolites in Human Brain Tumor Tissues by Chemometrics-Based Gas Chromatography-Mass Spectrometry Analysis for a Hypothetical Correlation between Pesticide Exposure and Risk Factor of Central Nervous System Tumors.

Pesticides are widely used, resulting in continuing human exposure with potential health impacts. Some exposures related to agricultural works have been associated with neurological disorders. Since the 2000s, the hypothesis of the role of pesticides in the occurrence of central nervous system (CNS) tumors has been better documented in the literature. However, the etiology of childhood brain cancers still remains largely unknown. The major objective of this work was to assess the potential role of pesticide exposure as a risk factor for CNS tumors based on questionnaires and statistical analysis of information collected from patients hospitalized in the Neurosurgery Department of the Habib Bourguiba Hospital Medium in Sfax, Tunisia, during the period from January 1, 2022, to May 31, 2023. It also aimed to develop a simple and rapid analytical method by the gas chromatography-mass spectrometry technique for the research traces of pesticide metabolites in some collected human brain tumor tissues in order to more emphasize our hypothesis for such a correlation between pesticide exposure and brain tumor development. Patients with a history of high-risk exposure were selected to conduct further analysis. Chemometric methods were adapted to discern intrinsic variation between pathological and control groups and ascertain effective separation with the identification of differentially expressed metabolites accountable for such variations. Three samples revealed traces of pesticide metabolites that were mostly detected at an early age. The histopathological diagnosis was medulloblastoma for a 10-year-old child and high-grade gliomas for 27- and 35-year-old adults. The bivariate analyses (odds ratio >1 and P value <5%) confirmed the great probability of developing cancer by an exposure case. The Cox proportional hazards model revealed the risk of carcinogenicity beyond the age of 50 as a long-term effect of pesticide toxicity. Our study supports the correlation between pesticide exposure and the risk of development of human brain tumors, suggesting that preconception pesticide exposure, and possibly exposure during pregnancy, is associated with an increased childhood brain tumor risk. This hypothesis was enhanced in identifying traces of metabolites from the carbamate insecticide class known for their neurotoxicity and others from pyridazinone, organochlorines (OCs), triazole fungicide, and N-nitroso compounds known for their carcinogenicity. The 2D-OXYBLOT analysis confirmed the neurotoxicity effect of insecticides to induce oxidative damage in CNS cells. Aldicarb was implicated in brain carcinogenicity confirmed by the identification of oxime metabolites in a stress degradation study. Revealing "aziridine" metabolites from the OC class may better emphasize the theory of detecting traces of pesticide metabolites at an early age. Overall, our findings lead to the recommendation of limiting the residential use of pesticides and the support of public health policies serving this objective that we need to be vigilant in the postmarketing surveillance of human health impacts.

Differential Proteome Profiling Analysis under Pesticide Stress by the Use of a Nano-UHPLC-MS/MS Untargeted Proteomic-Based Approach on a 3D-Developed Neurospheroid Model: Identification of Protein Interactions, Prognostic Biomarkers, and Potential Therapeutic Targets in Human IDH Mutant High-Grade Gliomas.

High-grade gliomas represent the most common group of infiltrative primary brain tumors in adults associated with high invasiveness, agressivity, and resistance to therapy, which highlights the need to develop potent drugs with novel mechanisms of action. The aim of this study is to reveal changes in proteome profiles under stressful conditions to identify prognostic biomarkers and altered apoptogenic pathways involved in the anticancer action of human isocitrate dehydrogenase (IDH) mutant high-grade gliomas. Our protocol consists first of a 3D in vitro developing neurospheroid model and then treatment by a pesticide mixture at relevant concentrations. Furthermore, we adopted an untargeted proteomic-based approach with high-resolution mass spectrometry for a comparative analysis of the differentially expressed proteins between treated and nontreated spheroids. Our analysis revealed that the majority of altered proteins were key members in glioma pathogenesis, implicated in the cellular metabolism, biological regulation, binding, and catalytic and structural activity and linked to many cascading regulatory pathways. Our finding revealed that grade-IV astrocytomas promote the downstream of the mitogen-activated-protein-kinases/extracellular-signal-regulated kinase (MAPK1/ERK2) pathway involving massive calcium influx. The gonadotrophin-releasing-hormone signaling enhances MAKP activity and may serve as a negative feedback compensating regulator. Thus, our study can pave the way for effective new therapeutic and diagnostic strategies to improve the overall survival.

Biotransformation of the Fluoroquinolone, Levofloxacin, by the White-Rot Fungus Coriolopsis gallica.

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.

Pistacia lentiscus L. Distilled Leaves as a Potential Cosmeceutical Ingredient: Phytochemical Characterization, Transdermal Diffusion, and Anti-Elastase and Anti-Tyrosinase Activities.

The present work was performed to investigate the phenolic composition of P. lentiscus L. distilled leaves (PDL) and examine its potential against certain key enzymes related to skin aging. High-pressure liquid chromatography coupled to mass spectrometry (HPLC-MS) and various separation procedures combined with nuclear magnetic resonance (NMR) and MS analysis were performed to isolate and identify compounds present in the ethyl acetate extract (EAE) of PDL. A high amount of flavonol glycoside was detected in EAE. Indeed, quercetin-3-O-rhamnoside (FC), myricetin-3-O-rhamnoside (FM2), and kaempferol-3-O-rhamnoside (FB2) were isolated from EAE, and are present in high quantities of 10.47 ± 0.26, 12.17 ± 0.74, and 4.53 ± 0.59 mg/g dry weight, respectively. A transdermal diffusion study was carried out to determine the EAE-molecules that may transmit the cutaneous barrier and showed that FM2 transmits the membrane barrier with a high amount followed by FC. EAE, FM2, and FC were tested against tyrosinase and elastase enzymes. Moreover, intracellular tyrosinase inhibition and cytotoxicity on skin melanoma cells (B16) were evaluated. The results indicated that EAE, FC, and FM2 have important inhibitory activities compared to the well-known standards, at non-cytotoxic concentrations. Therefore, they could be excellent agents for treating skin pigmentation and elasticity problems.

Effect of olive mill wastewaters on Scenedesmus sp. growth, metabolism and polyphenols removal.

BACKGROUND: The three-phase extraction process of olive oil produces highly contaminated wastewater (OMW). The elimination of this toxic by-product is an important environmental issue that requires the development of an appropriate management solution. The cultivation of microalgae using OMW as growth medium was therefore studied using single (the culture medium was formed by 0% to 80% ultrafiltered olive mill wastewater (OMUF) or OMW added to BG11) and two-stage strategies (microalgae were firstly cultivated in the BG11 medium. In the second stage, 40% and 80% of OMUF and OMW were added to the culture). In this work, biodegradation of OMW and subsequent extraction of lipid and antioxidant molecules was investigated as an ecofriendly method for the bioremediation and valorization of OMW. RESULTS: For two-stage cultivation, OMUF and OMW stress enhanced the intracellular amount of polyphenol accumulated in Scenedesmus sp. and exhibited the highest 2, 2-diphenyl-1- picrylhydrazyl radical (DPPH) and 2,2'-azino-bis (3-ethylbenzoline-6-sulfonate) radical (ABTS) scavenging ability compared with single-stage cultivation. Moreover, the lipid profile is dominated by polyunsaturated acids. In the single-stage cultivation, the Ch a, Ch b, carotenoid, carbohydrate and lipid content of 2.57, 7.4, 1.69, 368, and 644 g kg-1 were observed in 40% OMUF added culture, respectively, along with high biomass productivity and 58% of polyphenol removal. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the biomass of Scenedesmus sp. cultured on 40% OMUF did not show any toxic effect, making it an efficient strategy. CONCLUSION: The results indicate that Scenedesmus sp. is a promising microalga for the biotreatment of OMW and the extraction of bioactive metabolites. © 2021 Society of Chemical Industry.

Lipopeptides production by a newly Halomonas venusta strain: Characterization and biotechnological properties.

A halotolerant marine strain PHKT of Halomonas venusta was isolated from contaminated seawater as an efficient biosurfactant producer candidate, on low-value substrate (glycerol). The produced biosurfactants (Bios-PHKT) were characterized as lipopeptides molecules, belonging to surfactin and pumilacidin families, by using Thin Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FT-IR) and Tandem Mass Spectrometry (MALDI-TOF/MS-MS). Bios-PHKT has a critical micelle concentration (CMC) of 125 mg/L, and showed a high steadiness against a wide spectrum of salinity (0-120 g/L NaCl), temperature (4-121 °C) and pH (2-12), supporting its powerful tensioactive properties under various environmental conditions. Likewise, the cytotoxic test revealed that the biosurfactant Bios-PHKT, at concentrations lower than 125 µg/mL, was not cytotoxic for human HEK-293 cells since the cell survival is over than 80%. Furthermore, Bios-PHKT lipopeptides showed excellent anti-adhesive and anti-biofilm activities, being able to avoid and disrupt the biofilm formation by certain pathogenic microorganisms. In addition, the biosurfactant Bios-PHKT showed a remarkable anti-proliferative activity towards tumor B16 melanoma cell line. Besides, Bios-PHKT exhibited an excellent in vitro and in vivo wound healing process. In light of these promising findings, Bios-PHKT could be successfully used in different biotechnological applications.

A Novel Digestive α-Amylase from Blue Crab (Portunus segnis) Viscera: Purification, Biochemical Characterization and Application for the Improvement of Antioxidant Potential of Oat Flour.

This study reports on the purification and characterization of a digestive α-amylase from blue crab (Portunussegnis) viscera designated Blue Crab Amylase (BCA). The enzyme was purified to homogeneity by ultrafiltration, Sephadex G-100 gel filtration and Sepharose mono Q anion exchange chromatography, with the final purification fold of 424.02, specific activity of 1390.8 U mg-1 and 27.8% recovery. BCA, showing a molecular weight of approximately 45 kDa, possesses desirable biotechnological features, such as optimal temperature of 50 °C, interesting thermal stability which is enhanced in the presence of starch, high stability towards surfactants (Tween 20, Tween 80 and Triton X-100), high specific activity, quite high storage and broad pH range stability. The enzyme displayed Km and Vmax values, of 7.5 ± 0.25 mg mL-1 and 2000 ± 23 µmol min-1 mg-1 for potato starch, respectively. It hydrolyzed various carbohydrates and produced maltose, maltotriose and maltotetraose as the major end products of starch hydrolysis. In addition, the purified enzyme was successfully utilized for the improvement of the antioxidant potential of oat flour, which could be extended to other cereals. Interestingly, besides its suitability for application in different industrial sectors, especially food industries, the biochemical properties of BCA from the blue crab viscera provide novel features with other marine-derived enzymes and better understanding of the biodegradability of carbohydrates in marine environments, particularly in invasive alien crustaceans.

Effect of Mild Salinity Stress on the Growth, Fatty Acid and Carotenoid Compositions, and Biological Activities of the Thermal Freshwater Microalgae Scenedesmus sp.

Carotenoids have strong antioxidant activity as well as therapeutic value. Their production has been induced in algae under stressful culture conditions. However, the extreme culture conditions lead to the Programmed Cell Death (PCD) of algae, which affects their growth and productivity. This study was performed to evaluate the effect of salinity on the physiological and biochemical traits of Scenedesmus sp., thermal freshwater microalgae from Northern Tunisia. It was cultured under different NaCl concentrations ranging from 0 to 60 g/L. Results showed a good growth and high contents of total chlorophyll and carotenoids in Scenedesmus sp. cultured at 10 g/L of NaCl (salt-stressed 10 (Ss10)). The pigment composition of the Ss10 extract was acquired using HPLC-MS, and showed that the carotenoid fraction is particularly rich in xanthophylls. Moreover, the antioxidant (DPPH and FRAP) and enzymatic inhibition (tyrosinase and elastase) activities of the Ss10 extract were higher compared to those of the control culture. In addition, the cytotoxicity test on B16 cells showed that the Ss10 extract was non-toxic for all tested concentrations below 100 µg/mL. It also showed a rich unsaturated fatty acid (FA) composition. Therefore, these findings suggest that Scenedesmus sp. strain cultivated under mild stress salinity could be a source of biomolecules that have potential applications in the nutraceutical and cosmeceutical industries.

Contribution of Major Polyphenols to the Antioxidant Profile and Cytotoxic Activity of Olive Leaves.

AIMS: This study was designed to investigate the phytochemical profile and the cytotoxic activities of the eco-friendly extracts of olive leaves from Chemlali cultivar. MATERIALS AND METHODS: The Phenolic composition of olive leaves extracts, the antioxidant activity and the cytotoxic effects against MCF-7 and HepG2 cells were determined. RESULTS: Olive leaves extracts showed relevant total polyphenols contents. Oleuropein was the major detected phenolic compound reaching a concentration of 16.9 mg/ml. The antioxidant potential of the studied extracts varied from 23.7 to 46.5mM Trolox equivalents as revealed by DPPH and ABTS assays. Cytotoxicity experiments showed similar trends for both HepG2 and MCF-7 cells with the infusion extract being the most active. CONCLUSION: This study denotes that olive leaves may have great potential as endless bioresource of valuable bioactive compounds which may have a wide application.

Olive phenolic compounds attenuate deltamethrin-induced liver and kidney toxicity through regulating oxidative stress, inflammation and apoptosis.

The purpose of this study was to evaluate the protective effect of ethanolic olive fruit extract (OFE) and its phenolic compound, oleuropein (OLE), against hepato-renal toxicity induced by deltamethrin (DEM), a synthetic pyrethroid, in Wistar rats. The kidney and liver tissues were collected after 30 days of treatment for subsequent investigation. Rats that were given DEM had a highly significant elevation in the serum biomarkers as well as hepatic and renal levels of lipid peroxidation (MDA). Additionally, a significant reduction in the total antioxidant capacity (ABTS+), superoxide dismutase (SOD) and catalase (CAT) activities was noted. This toxic effect was confirmed by histological studies and the expression levels of inflammatory (cox-2) and apoptotic genes (bcl-2 and p53). The findings for the OFE and OLEtreated groups highlighted the efficacy of olive fruit phenolic compounds as hepatic and renal-protectant in DEM-induced hepato-renal toxicity through improving the oxidative status as well as suppressing inflammation and apoptosis. Therefore, they may be used as protective natural compounds against DEM-induced hepato-renal toxicity.

Assessment of Olea europaea L. fruit extracts: Phytochemical characterization and anticancer pathway investigation.

Olea europaea L. has been widely used as an advantageous rich source of bioactive compounds of high economic value leading to its use in pharmaceutical, cosmetic, and agriculture industries. Ethanolic extracts of olive fruits from three different cultivars (OFE) were studied for their phytochemical contents and were investigated for antioxidant activities and anticancer potential. Major polyphenols detected in these extracts were tyrosol, hydroxytyrosol, oleuropein, rutin, quercetin and glucoside forms of luteolin and apigenin. All these compounds have shown to significantly contribute to the antioxidant activity of OFE, which was evaluated by DPPH and ABTS assays. Proliferation of hepatic and colon cancer cells, HepG2 and Caco-2, were shown to be sensitive to OFE with IC50 less than 1.6mg/ml for all tested extracts. Moreover, flow cytometry analysis showed that OFE induced cell cycle arrest in the S-phase within both HepG2 and Caco-2 cells. This has triggered a cell death mechanism as shown by DNA fragmentation, expression of p53 and phosphorylation level of Akt and Erk proteins. Interestingly, these extracts could be further used as a potential source of natural compounds with both antioxidant and anticancer effects.

Oleuropein activated AMPK and induced insulin sensitivity in C2C12 muscle cells.

AIMS: Oleuropein has been recognized as an important medicinal compound because of its various biological properties, including anti-cancer, antidiabetic and anti-atherosclerotic activities. Here, we evaluate the antioxidant activity as well as the mechanism of the hypoglycemic effects of oleuropein in C2C12 cells and we establish the mechanism underlying these effects. MAIN METHODS: To perform this study, C2C12 cells viability was analyzed via MTT assay and the antioxidant activity was investigated by ROS and TBARS assays. Also, the effect of oleuropein on AMPK and PI3 kinase signaling pathways was evaluated. KEY FINDINGS: Treatment with oleuropein was able to protect cells against H2O2 induced stress in cells. On the other hand, the molecular bases of its actions have been scarcely understood. Oleuropein significantly enhanced glucose consumption and the phosphorylation of AMPK (AMP-activated protein kinase/ACC (acetyl-CoA carboxylase)) and MAPKs (mitogen-activated protein kinases), but not PI3 kinase (Phosphatidylinositol 3-kinase)/Akt. However, the co-treatment of oleuropein and insulin improved the insulin sensitivity via insulin-dependent (PI3 kinase/Akt) and insulin independent (AMPK/ACC) pathways. These results could be confirmed from the findings of GLUT4 translocation which was strongly enhanced in the case of oleuropein. SIGNIFICANCE: Our results provide important insights for the possible mechanism of action of oleuropein as a therapeutic agent in diabetic patients.

Biodegradation of malodorous thiols by a Brevibacillus sp. strain isolated from a Tunisian phosphate factory.

Hydrogen sulfide (H2S) and thiols (RSH) generated by the phosphate industry cause harmful effects on human health and quality of life. The present study aims to investigate and evaluate a bacterial strain CAT37 isolated from gas-washing wastewaters in terms of its properties and ability to degrade malodorous thiols. Gas-washing wastewater samples were submitted to physicochemical analyses and used for the isolation of thiol-degrading bacteria. The results from gas chromatography-mass spectrometry (GC-MS) analysis revealed that the isolated strain CAT37 was able to oxidize ∼99% of each thiol, decanethiol and dodecanethiol used as sole carbon and energy sources after 30 days of incubation at 37°C. The strain CAT37 displayed a biodegradative potential on several thiols known by their toxicity and odors. The results from phylogenetic and phenotypic analysis revealed that the CAT37 isolate belonged to the genus Brevibacillus, showing the highest sequence similarity to Brevibacillus agri. Overall, the results indicated that the strain CAT37 exhibited a number of attractive biodegradation abilities against thiols and could be considered a promising candidate for industrial application in future thiol biodeodorization strategies.