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Celecoxib (CXB) is a Biopharmaceutical Classification System (BCS) Class II molecule with high permeability that is practically insoluble in water. Because of the poor water solubility, there is a wide range of absorption and limited bioavailability following oral administration. These unfavorable properties can be improved using dry co-milling technology, which is an industrial applicable technology. The purpose of this study was to develop and optimize CXB nanoformulations prepared by dry co-milling technology, with a quality by design approach to maintain enhanced solubility, dissolution rate, and oral bioavailability. The resulting co-milled CXB composition using povidone (PVP), mannitol (MAN) and sodium lauryl sulfate (SLS) showed the maximum solubility and dissolution rate in physiologically relevant media. Potential risk factors were determined with an Ishikawa diagram, important risk factors were selected with Plackett-Burman experimental design, and CXB compositions were optimized with Central Composite design (CCD) and Bayesian optimization (BO). Physical characterization, intrinsic dissolution rate, solubility, and stability experiments were used to evaluate the optimized co-milled CXB compositions. Dissolution and permeability studies were carried out for the resulting CXB nanoformulation. Oral pharmacokinetic studies of the CXB nanoformulation and reference product were performed in rats. The results of in vitro and in vivo studies show that the CXB nanoformulations have enhanced solubility (over 4.8-fold (8.6 ± 1.06 µg/mL vs. 1.8 ± 0.33 µg/mL) in water when compared with celecoxib pure powder), and dissolution rate (at least 85% of celecoxib is dissolved in 20 min), and improved oral pharmacokinetic profile (the relative bioavailability was 145.2%, compared to that of Celebrex ® , and faster t max 3.80 ± 2.28 h vs. 6.00 ± 3.67 h, indicating a more rapid absorption rate).

Lacrimal gland plays a vital role in maintaining the health and function of the ocular surface. Dysfunction of the gland leads to disruption of ocular surface homeostasis and can lead to severe outcomes. Approaches evolving through regenerative medicine have recently gained importance to restore the function of the gland. Using human induced pluripotent stem cells (iPSCs), we generated functional in vitro lacrimal gland organoids by adopting the multi zonal ocular differentiation approach. We differentiated human iPSCs and confirmed commitment to neuro ectodermal lineage. Then we identified emergence of mesenchymal and epithelial lacrimal gland progenitor cells by the third week of differentiation. Differentiated progenitors underwent branching morphogenesis in the following weeks, typical of lacrimal gland development. We were able to confirm the presence of lacrimal gland specific acinar, ductal, and myoepithelial cells and structures during weeks 4-7. Further on, we demonstrated the role of miR-205 in regulation of the lacrimal gland organoid development by monitoring miR-205 and FGF10 mRNA levels throughout the differentiation process. In addition, we assessed the functionality of the organoids using the β -Hexosaminidase assay, confirming the secretory function of lacrimal organoids. Finally, metabolomics analysis revealed a shift from amino acid metabolism to lipid metabolism in differentiated organoids. These functional, tear proteins secreting human lacrimal gland organoids harbor a great potential for the improvement of existing treatment options of lacrimal gland dysfunction and can serve as a platform to study human lacrimal gland development and morphogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Asal, Koçak, Sarı, Reçber, Nemutlu, Utine and Güven.)

In this article, a serially connected dual column liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous separation and enantioseparation of proteinogenic amino acids. For this purpose, different achiral and chiral stationary phases (CSP) and mobile phase compositions have been tested. As a result of the optimization studies, the best enatioseparation for amino acids were achieved with a combination of zwitterionic and crown ether stationary phases using a gradient of two mobile phases: A (water:TFA 99.5:0.5, % v/v) and B (acetonitrile:ethanol:TFA 85:15:0.5, % v/v/v). The developed method provided simultaneous enantioseparation of all proteinogenic amino acids under this study including isomeric and isobaric ones except for proline. The method was successfully applied to human lung adenocarcinoma cells (A549) and healthy human lung epithelial cells (BEAS-2B) cultivated with d-amino acid containing cocktails in order to evaluate d-amino acids transfer rate in normal and cancer lines. Thed/l amino acid ratios were different in cancer and normal cell lines cultivated as mentioned above for aspartic acid, cysteine, methionine, phenylalanine, and serine., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Objectives: Non-alcoholic fatty liver disease (NAFLD) is one of the major causes of liver disease worldwide. Although various molecular mechanisms are effective in the initiation and progression, the exact pathway is not completely clarified. Recent findings suggest a role of the endocannabinoid system in the pathology of NAFLD. Inulin has been shown to be beneficial for NAFLD. With the first study, we investigated the effects of inulin supplementation on NAFLD via the endocannabinoid system in Wistar rats fed high-fat diet., Methods: Male Wistar rats were fed with control, control plus inulin, high-fat, and high-fat plus inulin diets for 12 wk. Inulin was added to diets in 15% weight/weight. Biochemical parameters, insulin, and adiponectin levels were determined. Steatosis, lobular inflammation, and total NAFLD activity scores (NAS) were determined by histopathological analysis and by magnetic resonance imaging. Anandamide and 2-arachidonylglycerol levels were measured by the liquid chromatography-tandem mass spectrometry method. Gene expression levels were determined by the quantitative polymerase chain reaction method., Results: Our results showed that the NAS of the high-fat diet was 4.16 ± 0.30, which was significantly higher than that of the other groups. Inulin decreased Homeostasis model assessment measuring insulin resistance (HOMA-IR), serum triacylglycerol, total cholesterol, and Aspartate aminotransferaselevels. Inulin also significantly decreased Cannabinoid receptor-1 and Patatin-like phospholipase-3 gene expressions in the liver. The 2-arachidonylglycerol levels in the liver were lower in the inulin-added groups. These effects of inulin were associated with NAS., Conclusions: Inulin prevented the development of NAFLD, possibly by affecting the expression of genes involved in the pathogenesis of NAFLD in the liver via endocannabinoids. The results of this study show that inulin may be a promising molecule in the treatment/prevention of NAFLD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material's adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

Aims: Endocannabinoids are biologically active cannabinoid-related substances endogenously synthesized in many mammalian tissues. Mainly two enzymes carry out their degradation; Fatty Acid Amide Hydrolase (FAAH) and Monoacylglycerol Lipase (MAGL). Endocannabinoids are shown to affect the modulation of inflammatory processes and airway responsiveness. In the present study, we investigated the effects of FAAH and MAGL inhibitor treatments in experimental allergic airway inflammation in guinea pigs., Materials and Methods: Guinea pigs were sensitized and challenged by ovalbumin to induce an allergic asthma model. Then, the effects of FAAH inhibitor URB597, MAGL inhibitor JZL184, and dual (FAAH/MAGL) inhibitor JZL195 on airway inflammation and hyperreactivity were evaluated., Key Findings: Ovalbumin challenge increased airway reactivity, IgE in serum, IL-4, and IL-13, and the percentage of eosinophils in bronchoalveolar lavage (BAL). In addition, inhibition of FAAH or MAGL enzymes leads to an increase in endocannabinoid levels. The selective inhibition of the FAAH enzyme prevented inflammation indicators such as cytokine production and inflammatory cell infiltration but had a negligible effect on airway hyperreactivity. However, the inhibition of the MAGL enzyme or dual inhibition of both FAAH and MAGL enzymes tent to moderate both pulmonary inflammation and airway hyperreactivity., Significance: We have previously demonstrated that modulation of endocannabinoid levels in the airways by FAAH or MAGL inhibition can be useful in preventing acute lung inflammation. The results of the present study further suggest that FAAH and MAGL inhibitor treatment can also be a promising strategy for bronchial hyperreactivity and airway inflammation in allergic asthma., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Recent research effort in biomaterial development has largely focused on engineering bio-instructive materials to stimulate specific cell signaling. Assessing the biological performance of these materials using time-consuming and trial-and-error traditional low-throughput screening techniques remains a critical challenge in the field. In contrast, the use of increasingly sophisticated omics technologies to facilitate high-throughput screening of unbiased global understanding of cell-biomaterial interactions at gene, epigenetic, mRNA, protein, metabolite, and lipid levels holds great potential to predict the therapeutic outcome of biomaterials with specific properties. In this review, we highlight the potential use of omics technologies - namely transcriptomics, proteomics, metabolomics and lipidomics - in biomaterial design and deciphering of the fundamental cell behaviors ( e.g. , adhesion, migration, differentiation) in response to cell-biomaterial interactions. Moreover, the potential challenges and prospects of high-throughput analysis platforms are discussed rationally, providing an insight into the developing field and its use in biomaterials science.

Swallowing oral solid dosage forms is challenging in patients with dysphagia who are at risk of aspiration or choking. The most common method to facilitate drug administration in dysphagia patients is to mix the powdered drug with a small amount of thickened water, however little is known about the effects of this method on in vivo bioavailability of drugs. This study aimed to evaluate the impact of thickened liquids on dissolution rate and bioavailability of levetiracetam as a model drug. Powdered commercial tablets of levetiracetam, carbamazepine, atenolol and cefixime were mixed with water thickened with two commercial thickeners, modified maize starch (MS) and xanthan gam (XG), at three thickness levels: nectar, honey and pudding in test groups, and mixed with only water in the control group. At the first stage, the effects of thickened water on in vitro drug release of 4 drugs (levetiracetam, carbamazepine, atenolol and cefixime) were tested by using dialysis membrane method. Addition of both thickeners significantly reduced the release of three drugs compared to the control group, except carbamazepine. Levetiracetam which had the highest solubility was chosen as the model drug for in vivo experiments. In the second stage, New Zealand albino female rabbits (n=24) were divided into two groups as: control group (water+drug, n=6) and test group (thickened water+drug, n=18). Powdered levetiracetam tablets were mixed with water thickened with XG (n=9, 1.2%, 2.4%, 3.6%) and MS (n=9, 4%, 6%, 8%) at three thickness levels and administered to the rabbits by intragastric gavage. Blood samples were collected at 9 time points following administration. After two-weeks of wash-out, test groups were crossed over and sample collection was repeated. Blood samples were analysed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). An in vitro-in vivo correlation (IVIVC) model was developed using in vitro drug dissolution (%) and in vivo plasma concentrations of levetiracetam for control group and test groups. The peak plasma concentration (C max ) was lower and time to reach C max (t max ) was relatively higher in test groups compared to control group. The lowest C max was detected at the highest thickness level, however, the differences between groups were not statistically significant (p=0.117 and p=0.495 for C max and t max , respectively). No significant difference in total amount of levetiracetam absorbed (AUC) was found between groups (p=0.215 and p=0.183 for AUC infinity and AUC last , respectively). The comparisons according to the type of thickener also revealed that pharmacokinetic parameters did not significantly differ between groups, except for a significantly lower C max when drug was mixed with MS-thickened water at nectar consistency (1.2%) compared to drug mixed with XG (4%) at the same thickness level (p=0.038). A good correlation was observed between in vitro and in vivo data, which was characterized by higher r 2 values as the concentration of the thickening agents was increased, but not for all thickness levels studied, indicating an inability of this in vitro model to fully predict the in vivo response. These results suggest that regardless of the thickness level, the administration of levetiracetam with two commercial thickening agents commonly used in dysphagia for safe swallowing, do not affect the pharmacokinetic efficiency and thus, the bioavailability of the drug., (Copyright © 2022. Published by Elsevier B.V.)

Aims: The relationship between type 2 diabetes and Alzheimer's disease (AD) provides evidence that insulin and insulin sensitizers may be beneficial for the treatment of AD. The present study investigated the effect and mechanism of action of intranasal metformin treatment on impaired cognitive functions in an experimental mice model of AD., Main Methods: Intracerebroventricularly (ICV) streptozotocin (STZ)-injected mice were treated with intranasal or oral metformin for 4 weeks. Learning and memory functions were evaluated using Morris water maze. Metformin and Aβ42 concentrations were determined by liquid chromatography tandem mass spectrometry and ELISA respectively. The expressions of insulin receptor, Akt and their phosphorylated forms were determined in the hippocampi and cerebral cortices of mice., Key Findings: ICV-STZ-induced AD mice displayed impaired learning and memory functions which were improved by metformin treatment. ICV-STZ injection or intranasal/oral metformin treatments had no effect on blood glucose concentrations. Intranasal treatment yielded higher concentration of metformin in the hippocampus and lower in the plasma compared to oral treatment. ICV-STZ injection and metformin treatments did not change amyloid β-42 concentration in the hippocampus of mice. In hippocampal and cortical tissues of ICV-STZ-induced AD mice, insulin receptor (IR) and Akt expressions were unchanged, while phosphorylated insulin receptor (pIR) and pAkt expressions decreased compared to control. Metformin treatments did not change IR and Akt expressions but increased pIR and pAkt expressions., Significance: The present study showed for the first time that intranasal metformin treatment improved the impaired cognitive functions through increasing insulin sensitivity in ICV-STZ-induced mice model of AD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration., (© 2022 Wiley-VCH GmbH.)

Oxidative stress may contribute to the development of schizophrenia and antipsychotics used in schizophrenia treatment may also cause oxidative stress. Gene polymorphisms on antioxidant and repair enzymes are responsible for individual variations and may change the efficacy of olanzapine treatment among schizophrenia patients. In our study, we assessed oxidative stress-related metabolite changes due to genetic polymorphisms on first diagnosed-schizophrenia patients treated with olanzapine. Blood samples ( n  = 30 patients) were taken before treatment (T1), after 10 ± 1 days (T2), and after 3 ± 1 months (T3). T1 served as control for T2 and T3, since it is advantageous to perform on same patient to evaluate the impact of olanzapine only. GSTs ( GSTM1 , GSTT1 , and GSTP1 ) and OGG1 gene polymorphisms were analyzed by polymerase chain reaction. Changes in metabolites were detected with metabolomics profiling by gas chromatography-mass spectrometry according to each genotype before and after treatment. Multivariate analysis showed that metabolomics profiles differed after olanzapine treatment regardless gene polymorphisms. Tryptophan could be a biomarker in response to olanzapine treatment since its levels were increased after treatment. GSTM1 gene polymorphism caused significant changes in some metabolites after treatment. Urea, palmitic acid, and caprylic acid levels increased and alanine levels decreased in patients with GSTM1 null genotypes after olanzapine. In future, targeted metabolomics with these prominent metabolites and assessing gene expressions of GSTs will be beneficial to understand the mechanism of action., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Antiviral drugs have gained much more attention in recent years due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and many drug candidates are currently under investigation in order to end pandemic. Molnupiravir, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, is one of the promising candidates for SARS-CoV-2 treatment. In this study, a RP-HPLC method was developed for the determination of Molnupiravir and applied for in vitro permeability studies of self-emulsifying drug delivery system (SEDDS) formulations using Caco-2 cell line. Discovery® HS C18 Column (75 ×4.6 mm, 3 µm) was used at 30 °C. Isocratic elution was performed with ACN:water (20:80 v/v) mixture. The flow rate was 0.5 mL/min and UV detection was at 240 nm. Molnupiravir eluted within 5 min. Molnupiravir was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions. Peak homogeneity data of Molnupiravir in the stressed samples peak obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The developed method was validated according to the International Council for Harmonisation (ICH) guidelines and found to be linear within the range 0.1-60.0 μg/mL. The method was simple, rapid, selective, sensitive, accurate, precise, robust and rugged. Thus, it was applied successfully for permeability quantitation of Molnupiravir in nanoformulations. The apparent permeability of Molnupiravir in SEDDS formulations, which have droplet size under 350 nm, was calculated as 3.20 ± 0.44 × 10 -6 cm/s., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: This study aimed to examine patients with lead poisoning in terms of metabolomic profiles and bioactive lipids (oxysterols and sphingosine 1-phosphate [S1P]) before and after chelation therapy., Methods: Consent was obtained from 42 individuals diagnosed with lead poisoning and blood and urine samples were collected before and after chelation therapy. The levels of 7-ketocholesterol (7-KC), cholestan-3b,5a,6b-triol (Ctriol), and S1P were measured via LC-MS/MS. Metabolomic analysis was performed via GC-MS., Results: 7-KC and C-triol levels were detected higher before chelation therapy compared with after therapy (P < 0.001 for both). S1P levels were measured higher before the therapy. The results also showed that sphingolipid metabolism-related pathways were affected by lead toxicity as well as other related pathways., Conclusion: This preliminary study showed that lipid metabolism is affected in lead exposure and chelation therapy is effective in reversing possible damage., Competing Interests: Conflict of interest: All the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 American College of Occupational and Environmental Medicine.)

To design and develop K-RAS silencing small interfering RNA (siRNA)-loaded poly (D, L-lactic-co-glycolic acid) nanoparticles and evaluate their efficacy in the treatment of K-RAS mutant lung cancer. The nanoparticles prepared by the double emulsion solvent evaporation method were characterized by TEM, FTIR and XPS analyzes and evaluated in vitro by XTT, PCR, ELISA, and Western-Blot. Metabolomic analyzes were performed to evaluate the changes in metabolic profiles of the cells after nanoparticles treatment. The nanoparticles were obtained with a particle size less than 250 nm, a polydispersity index around 0.1, a surface charge of (-12) - (+14) mV, and 80% of the siRNA encapsulation. The nanoparticles didn't affect cell viability of the cells after 72 hours. In cancer cells, KRAS expression was decreased by up to 50%, protein levels were decreased by more than 90%. The formulated siRNA delivery nanoparticles can be promising treatment in lung cancer.

Coenzyme Q 10 (CoQ 10 ) deficiency exhibits signs of multiple organ dysfunctions, particular subtypes present isolated kidney involvement progressing to chronic kidney disease. In these patients, early administration of oral CoQ 10 has been shown to decrease proteinuria and to delay development of chronic kidney disease, which suggests that it may have a renoprotective potential in these patients. However, CoQ 10 bioavailability in mitochondria is low, therefore its efficacy is limited. We aimed to develop mitochondria-targeted CoQ 10 loaded poly(lactic-co-glycolic acid)-poly(ethylene glycol)-triphenylphosphonium nanoparticles (CoQ 10 -TPP-NPs) that would be more efficient in the treatment of CoQ 10 nephropathies. These nanoparticles were found to have a size of approximately 150 nm and a zeta potential of + 20 mV. The entrapment efficiency of the nanoparticles was determined as 40%. Cytotoxicity studies showed no effect on the viability of the human kidney proximal tubule epithelial cells exposed to the nanoparticles. The efficacy of the formulated nanoparticles on in vitro disease model, which was developed in the human kidney proximal tubule epithelial cells by siRNA based silencing of the COQ8B, was evaluated through mitochondrial functions by means of metabolomic analyses. We showed that the treatment of COQ8B -/- cells with mitochondria-targeted nanoparticles was more effective in increasing the tricarboxylic acid cycle rate compared to free-CoQ 10 . Our formulation would be more effective in treatment of CoQ 10 -related nephropathies than conventional formulations., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The level of the major endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are altered in several types of carcinomas, and are known to regulate tumor growth. Thusly, this study hypothesized that the HEp-2 human laryngeal squamous cell carcinoma (LSCC) cell line releases AEA and 2-AG, and aimed to determine if their exogenous supplementation has an anti-proliferative effect in vitro. In this in vitro observational study a commercial human LSCC cell line (HEp-2) was used to test for endogenous AEA and 2-AG release via liquid chromatography-tandem mass spectrometry (LC-MS/MS). The anti-proliferative effect of AEA and 2-AG supplementation was evaluated via WST-1 proliferation assay. It was observed that the HEp-2 LSCC cell line released AEA and 2-AG; the median quantity of AEA released was 15.69 ng mL -1 (range: 14.55-15.95 ng mL -1 ) and the median quantity of 2-AG released was 2.72 ng -1 (range: 2.67-2.74 ng mL -1 ). Additionally, both AEA and 2-AG exhibited an anti-proliferative effect. The anti-proliferative effect of 2-AG was stronger than that of AEA. These findings suggest that AEA might function via a CB1 receptor-independent pathway and that 2-AG might function via a CB2-dependent pathway. The present findings show that the HEp-2 LSCC cell line releases the major endocannabinoids AEA and 2-AG, and that their supplementation inhibits tumor cell proliferation in vitro. Thus, cannabinoid ligands might represent novel drug candidates for laryngeal cancers, although functional in vivo studies are required in order to validate their potency., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: Breast cancer is the most common cancer in women and is the second most common cause of cancer related mortality. Metabolomics, the identification of small metabolites, is a technique for determining the amount of these metabolites. Objectives: This study aimed to identify markers for the early diagnosis of brain metastasis by metabolomic methods in breast cancer patients . Methods: A total of 88 breast cancer patients with distant metastases were included in the study. The patients were divided into two groups according to their metastasis status: patients with brain metastases and distant metastases without any brain metastases. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) and gas chromatography-mass spectrometry (GC-MS) analysis methods were used for metabolomic analyses. Results: 33 of them, 88 patients had brain metastasis, and 55 patients had distant metastases without brain metastasis. A total of 72 and 35 metabolites were identified by the GC-MS and LC-qTOF-MS analysis, respectively. 47 of them were found to be significantly different in patients with brain metastasis. The pathway analysis, performed with significantly altered metabolites, showed that aminoacyl tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, alanine, aspartate, and glutamate metabolism, arginine biosynthesis, glycine, serine, and threonine metabolism pathways significantly altered in patients with brain metastasis. Predictive accuracies for have identifying the brain metastasis were performed with receiver operating characteristic (ROC) analysis, and the model with fifteen metabolites has 96.9% accuracy. Conclusions: While these results should be supported by prospective studies, these data are promising for early detection of brain metastasis with markers in liquid biopsy samples.

The contribution of the endocannabinoid system to both physiology and pathological processes in the respiratory system makes it a promising target for inflammatory airway diseases. Previously, we have shown that increasing the tissue endocannabinoid levels by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) inhibitors can prevent airway inflammation and hyperreactivity. In this study, the changes in the levels of major metabolites of endocannabinoids by systemic and local FAAH or MAGL inhibitor treatments were evaluated. Mice were treated with either the FAAH inhibitor URB597 or the MAGL inhibitor JZL184 by local (intranasal) or systemic (intraperitoneal) application. Bronchoalveolar lavage (BAL) fluids and lungs were isolated afterward in order to perform histopathological and metabolomic analyses. There were no significant histopathological changes in the lungs and neutrophil, and macrophage and lymphocyte numbers in BAL fluid were not altered after local and systemic treatments. However, GC-MS-based metabolomics profile allowed us to identify 102 metabolites in lung samples, among which levels of 75 metabolites were significantly different from the control. The metabolites whose levels were changed by treatments were mostly related to the endocannabinoid system and energy metabolism. Therefore, these changes may contribute to the anti-inflammatory effects of URB597 and JZL184 treatments in mice., (© 2021 John Wiley & Sons, Ltd.)

Self-assembling bio-instructive materials that can provide a biomimetic tissue microenvironment with the capability to regulate cellular behaviors represent an attractive platform in regenerative medicine. Herein, we develop a hybrid neuro-instructive hydrogel that combines the properties of a photo-crosslinkable gelatin methacrylate (GelMA) and self-assembling peptide amphiphiles (PAs) bearing a laminin-derived neuro-inductive epitope (PA-GSR). Electrostatic interaction and ultraviolet light crosslinking mechanisms were combined to create dual-crosslinked hybrid hydrogels with tunable stiffness. Spectroscopic, microscopic and theoretical techniques show that the cationic PA-GSR(+) electrostatically co-assembles with the negatively charged GelMA to create weak hydrogels with hierarchically ordered microstructures, which were further photo-crosslinked to create mechanically robust hydrogels. Dynamic oscillatory rheology and micromechanical testing show that photo-crosslinking of the co-assembled GelMA and PA-GSR(+) hydrogel results in robust hydrogels displaying improved stiffness. Gene expression analysis was used to show that GelMA/PA-GSR(+) hydrogels can induce human mesenchymal stem cells (hMSCs) into neural-lineage cells and supports neural-lineage specification of neuroblast-like cells (SH-SY5Y) in a growth-factor-free manner. Also, metabolomics analysis suggests that the hydrogel alters the metabolite profiles in the cells by affecting multiple molecular pathways. This work highlights a new approach for the design of PA-based hybrid hydrogels with robust mechanical properties and biological functionalities for nerve tissue regeneration.

Roemerine is a naturally occurring aporphine alkaloid. In this study, we screened a conformer library of Food and Drug Administration (FDA)-approved drugs to identify similar drugs that can assist in identifying the biological targets of roemerine. To assess the neuroactivity in vitro , we measured the levels of cell metabolites, Brain-Derived Neurotrophic Factor (BDNF) and serotonin (5-HT) in SH-SY5Y cell line. By means of structure-based virtual screening, we identified five drugs that are similar to roemerine; mirtazapine, atomoxetine, epinastine, diphenhydramine and orphenadrine. GC-MS metabolomics study revealed that roemerine has a high impact on alanine-aspartate-glutamate pathway in cell lysate and cultured medium. Additionally, roemerine increased intercellular 5-HT level and intracellular BDNF protein expression at 10 µM. In conclusion, roemerine - a major alkaloid in antidepressant-like effect possessing plants ( P. lacerum and P. syriacum ) - has a neuronal activity through increasing BDNF protein expression and affecting serotonergic and glutamatergic systems in SH-SY5Y cell line.

Decellularization of extracellular matrices offers an alternative source of regenerative biomaterials that preserve biochemical structure and matrix components of native tissues. In this study, decellularized bovine spinal cord meninges (dSCM)-derived extracellular matrix hydrogel (MeninGEL) is fabricated by employing a protocol that involves physical, chemical, and enzymatic processing of spinal meninges tissue and preserves the biochemical structure of meninges. The success of decellularization is characterized by measuring the contents of residual DNA, glycosaminoglycans, and hydroxyproline, while a proteomics analysis is applied to reveal the composition of MeninGEL. Frequency and temperature sweep rheometry show that dSCM forms self-supporting hydrogel at physiological temperature. The MeninGEL possesses excellent cytocompatibility. Moreover, it is evidenced with immuno/histochemistry and gene expression studies that the hydrogel induces growth-factor free differentiation of human mesenchymal stem cells into neural-lineage cells. Furthermore, MeninGEL instructs human umbilical vein endothelial cells to form vascular branching. With its innate bioactivity and low batch-to-batch variation property, the MeninGEL has the potential to be an off-the-shelf product in nerve tissue regeneration and restoration., (© 2021 John Wiley & Sons Ltd.)

Vitreous humor (VH) is not considered as a critical structure in the radiotherapy planning process. In the present study, an experimental animal model was performed to examine the effects of radiotherapy on VH. The right eyes of twelve New Zealand rabbits were irradiated to 60 Gy in 3 fractions in accordance with the scheme used in the treatment of uveal melanoma in our clinic, and contralateral (left) eyes were considered as control. Weekly ophthalmologic examination was performed after irradiation, for three months. At the end of the third month, enucleation and vitreous collection were conducted. The vitreous samples were subjected to metabolomic analyses, ELISA analyses, viscosity measurements, and electron microscopic examination. In control and experimental vitreous samples, 275 different metabolites were identified, and 34 were found to differ significantly between groups. In multivariate analyzes, a clear distinction was observed between control and irradiated vitreous samples. Pathway analysis revealed that nine pathways were affected, and these pathways were mainly related to amino acid metabolism. A significant decrease was observed in the expressions of type II, V, and XI collagens in protein level in the ELISA. There was a non-significant decrease in type IX collagen and viscosity. Electron microscopic examination revealed disrupted collagen fibrillar ultra-structure and dispersed collagen fragments in the experimental vitreous. An intact vitreous is essential for a healthy eye. In this study, we observed that radiation causes changes in the vitreous that may have long-term consequences., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Objectives: This study aimed to determine the effect of Enterococcus faecalis on the cell growth and hyphal formation of Candida albicans and to understand the exact mechanism of candidal inhibition by the existence of E. faecalis by metabolomic analysis., Materials and Methods: Single- and dual-species biofilms of E. faecalis and C. albicans were formed in a microtiter plate, and the metabolomic profiles of both biofilms was determined by gas chromatography-mass spectrometry. The hyphal cell growth of C. albicans after treatment with both the supernatant and biofilm cells of E. faecalis was examined microscopically. The expression levels of Efg1 and the images of C. albicans cell wall in single- and dual-species biofilms were determined by real-time quantitative polymerase chain reaction and transmission electron microscopy, respectively. The violacein levels produced by Chromobacterium violaceum were measured to determine the quorum sensing (QS) inhibitory activity of single- and dual-species biofilms., Results: The biofilm cell growth, Efg1 expression, and hyphal development of C. albicans were inhibited by E. faecalis . Compared to single-species biofilms, alterations in carbohydrate, amino acid, and polyamine metabolites were observed in the dual-species biofilm for both microorganisms. Putrescine and pipecolic acid were detected at high levels in dual-species biofilm. A thicker β-glucan chitin and a denser and narrower fibrillar mannan layer of C. albicans cell wall were observed in dual-species biofilm. QS inhibitory activity was higher in dual-species biofilm suspensions of E. faecalis and C. albicans than in their single-species biofilms., Conclusion: E. faecalis inhibited the hyphal development and biofilm formation of C. albicans . Biofilm suspensions of C. albicans and E. faecalis showed an anti-QS activity, which increased even further in the environment where the two species coexisted. Investigation of putrescine and pipecolic acid can be an important step to understand the inhibition of C. albicans by bacteria.

Globally, simvastatin is one of the most commonly used statin drugs. Its antimicrobial properties have been investigated against various pathogens. However, its effect on biological processes in bacteria has been unclear. This study focused on altered biological and metabolic processes at protein and metabolite levels induced by simvastatin. MS-based proteomics and metabolomics were used to investigate the altered proteins and metabolites between experimental groups. Proteomics results showed that simvastatin induced various antimicrobial targets such as chaperon protein DnaK and cell division protein FtsZ. Metabolomics results revealed phenotypic changes in cells under simvastatin stress. Integrated proteomics and metabolomics result indicated that various metabolic processes were altered to adapt to stress conditions. Energy metabolism (glycolysis, tricarboxylic acid cycle, etc.), amino acid synthesis and ribosomal proteins, and purine and pyrimidine synthesis were induced by the effect of simvastatin. This study will contribute to the understanding of antimicrobial properties of statin drugs., (© 2021 John Wiley & Sons, Ltd.)

Megaconial Congenital Muscular Dystrophy (CMD) is a rare autosomal recessive disorder characterized by enlarged mitochondria located mainly at the periphery of muscle fibers and caused by mutations in the Choline Kinase Beta (CHKB) gene. Although the pathogenesis of this disease is not well understood, there is accumulating evidence for the presence of mitochondrial dysfunction. In this study, we aimed to investigate whether imbalanced mitochondrial dynamics affects mitochondrial function and bioenergetic efficiency in skeletal muscle cells of Megaconial CMD. Immunofluorescence, confocal and transmission electron microscopy studies revealed impaired mitochondrial network, morphology, and localization in primary skeletal muscle cells of Megaconial CMD. The organelle disruption was specific only to skeletal muscle cells grown in culture. The expression levels of mitochondrial fission proteins (DRP1, MFF, FIS1) were found to be decreased significantly in both primary skeletal muscle cells and tissue sections of Megaconial CMD by Western blotting and/or immunofluorescence analysis. The metabolomic and fluxomic analysis, which were performed in Megaconial CMD for the first time, revealed decreased levels of phosphonucleotides, Krebs cycle intermediates, ATP, and altered energy metabolism pathways. Our results indicate that reduced mitochondrial fission and altered mitochondrial energy metabolism contribute to mitochondrial dysmorphology and dysfunction in the pathogenesis of Megaconial CMD., (© 2021. The Author(s).)

Cystic Echinococcosis (CE) is one of the life-threatening diseases worldwide. It is a parasitic zoonosis caused by tapeworms of the species Echinococcus granulosus sensu lato (s.l). The treatment options of CE vary from simple "watch and wait" approach to invasive treatment, based on the type and especially the nature of the cyst (active/inactive). Serological tests are inadequate to distinguish between active and inactive CE. A diagnostic reference that can determine whether the cyst is active or inactive can easily guide the treatment strategy. We aimed to test whether gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadropole time of flight mass spectrometry (LC-qTOF-MS) based metabolomics can establish a plasma metabolic fingerprint of CE patients and identify a diagnostic reference to discriminate active and inactive CE cysts. Metabolite concentrations were measured in plasma samples of 36 active CE patients, 17 inactive CE patients and 31 healthy controls. Multivariate statistical analysis on 232 identified metabolites obtained from two analytical platforms was performed by using principle component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) methods. The PLS-DA scores plot of the combined data set demonstrated a good separation between the groups. Compared to the healthy control group, decreased levels of squalene and increased levels of glyceric acid, 3-phosphoglycerate, glutamic acid, palmitoleic acid and oleic acid were determined in the CE patients. However, decreased levels of 3-phosphoglycerate and increased levels of 4-hydroxyphenylacetylglutamine, docosahexanoic acid were determined in active CE patients compared to the inactive CE patients. Determination of differences in metabolites may provide detailed understandings of potential metabolic process associated with active and inactive CE patients, and altered specific metabolic changes may provide some clues to obtain diagnostic reference for CE. This study has certain limitations: a. various factors affecting results of metabolomic studies such as lifestyle and dietary habits of the patients could not be fully controlled b. other infectious or malignant diseases of the liver should also be included as a positive control to evaluate the specificity of the diagnostic references., (Copyright © 2021. Published by Elsevier B.V.)

Background: Aerosol therapy is commonly used by intensivists during invasive mechanical ventilation. More information is needed to optimize outcomes. The first aim of this study was to assess the deposition of salbutamol on components of a closed mechanical ventilation system, both in the presence and absence of biofilm generated by Acinetobacter baumannii. The second aim was to evaluate the deposition of salbutamol, using a single dose and a double dose, delivered via a jet nebulizer placed between the flexible tube and the heat and moisture exchanger., Methods: A mechanical ventilator was connected to a standard system, and a jet nebulizer was placed between the heat and moisture exchanger and the flexible tube. Clinical isolates of A. baumanii were used to generate a biofilm layer on the endotracheal tube. Two amounts of salbutamol were delivered via the jet nebulizer. An analytical liquid chromatography tandem mass spectrometry method was developed to evaluate salbutamol deposition., Results: The presence of a biofilm on the endotracheal tube had no impact on salbutamol deposition ( P = .83). There was no difference in surface deposition of salbutamol on component parts of the closed system in a comparison of a single dose and a double dose delivered via a jet nebulizer., Conclusions: Our findings indicate that an A. baumannii biofilm had no impact on the extent of salbutamol deposition. Salbutamol deposition was comparatively low and could be delivered without removal of the heat and moisture exchanger., (Copyright © 2021 by Daedalus Enterprises.)

New technologies permit determining metabolomic profiles of human diseases by fingerprinting metabolites levels. However, to fully understand metabolomic phenotypes, metabolite levels and turnover rates are necessary to know. Krebs cycle is the major hub of energy metabolism and cell signaling. Traditionally, 13 C stable isotope labeled substrates were used to track the carbon turnover rates in Krebs cycle metabolites. In this study, for the first time we introduce H 2 [ 18 O] based stable isotope marker that permit tracking oxygen exchange rates in separate segments of Krebs cycle. The chromatographic and non-chromatographic parameters were systematically tested on the effect of labeling ratio of Krebs cycle mediators to increase selectivity and sensitivity of the method. We have developed a rapid, precise, and robust GC-MS method for determining the percentage of 18 O incorporation to Krebs cycle metabolites. The developed method was applied to track the cancer-induced shift in the Krebs cycle dynamics of Caco-2 cells as compared to the control FHC cells revealing Warburg effects in Caco-2 cells. We demonstrate that unique information could be obtained using this newly developed 18 O-labeling analytical technology by following the oxygen exchange rates of Krebs cycle metabolites. Thus, 18 O-labeling of Krebs cycle metabolites expands the arsenal of techniques for monitoring the dynamics of cellular metabolism. Moreover, the developed method will allow to apply the 18 O-labeling technique to numerous other metabolic pathways where oxygen exchange with water takes place., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Alternative anti-biofilm agents are needed to combat Pseudomonas aeruginosa infections. The mechanisms behind these new agents also need to be revealed at a molecular level., Materials and Methods: The anti-biofilm effects of 10 plant-derived compounds on P. aeruginosa biofilms were investigated using minimum biofilm eradication concentration (MBEC) and virulence assays. The effects of ciprofloxacin and compound combinations on P. aeruginosa in mono and triple biofilms were compared. A metabolomic approach and qRT-PCR were applied to the biofilms treated with ciprofloxacin in combination with baicalein, esculin hydrate, curcumin, and cinnamaldehyde at sub-minimal biofilm inhibitory concentration (MBIC) concentrations to highlight the specific metabolic shifts between the biofilms and to determine the quorum sensing gene expressions, respectively., Results: The combinations of ciprofloxacin with curcumin, baicalein, esculetin, and cinnamaldehyde showed more reduced MBICs than ciprofloxacin alone. The quorum sensing genes were downregulated in the presence of curcumin and cinnamaldehyde, while upregulated in the presence of baicalein and esculin hydrate rather than for ciprofloxacin alone. The combinations exhibited different killing effects on P. aeruginosa in mono and triple biofilms without affecting its virulence. The findings of the decreased metabolite levels related to pyrimidine and lipopolysaccharide synthesis and to down-regulated alginate and lasI expressions strongly indicate the role of multifactorial mechanisms for curcumin-mediated P. aeruginosa growth inhibition., Conclusions: The use of curcumin, baicalein, esculetin, and cinnamaldehyde with ciprofloxacin will help fight against P. aeruginosa biofilms. To the best of our knowledge, this is the first study of its kind to define the effect of plant-based compounds as possible anti-biofilm agents with low MBICs for the treatment of P. aeruginosa biofilms through metabolomic pathways.

Bone marrow (BM) niches are special microenvironments that work in harmony with each other for the regulation and maintenance of hematopoiesis. Niche investigations have thus far been limited to various model organisms and animal studies; therefore, little is known about different niches in healthy humans. In this study, a special harvesting method for the collection of BM from two different anatomical regions in the iliac crest of humans was used to investigate the presence of different niches in BM. Additionally, metabolomic and transcriptomic profiles were compiled using comparative 'omics' technologies, and the main cellular pathways and corresponding transcripts and metabolites were identified. As a result, we found that the energy metabolism between the regions was different. This study provides basic broad data for regenerative medicine in terms of the design of the appropriate microenvironment for in vitro hematopoietic niche modeling, and identifies the normal reference values that can be compared in hematological disease., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Aim: The assessment of efficacy should be paralleled with extensive pharmacokinetic parameters, and a valid bioanalytical method is a pre-condition for accurate plasma concentration. Materials & methods: A simple, specific, rapid and sensitive LC-MS/MS method has been developed for quantitative analysis of aprepitant in rat plasma. A C18 column was used as stationary phase and the mobile phase consisted of a mixture of formic acid in water and formic acid in acetonitrile. Quantification was performed using multiple reaction monitoring mode. Results: The selectivity, linearity, accuracy, precision, robustness and ruggedness of the method were evaluated in accordance with bioanalytical method validation guideline of ICH and all results were within the acceptable range. Conclusion: The validated LC-MS/MS method was found to be useful for the quantitative analysis of aprepitant in rat plasma samples.

Therapeutic agents used for non-small cell lung cancer (NSCLC) have limited curative efficacy and may trigger serious adverse effects. Cannabinoid ligands exert antiproliferative effect and induce apoptosis on numerous epithelial cancers. We confirmed that CB1 receptor (CB1R) is expressed in NSCLC cells in this study. Arachidonoylcyclopropylamide (ACPA) as a synthetic, CB1R-specific ligand decreased proliferation rate in NSCLC cells by WST-1 analysis and real-time proliferation assay (RTCA). The half-maximal inhibitory concentration (IC50) dose of ACPA was calculated as 1.39 × 10 -12  M. CB1 antagonist AM281 inhibited the antiproliferative effect of ACPA. Flow cytometry and ultrastructural analyzes revealed significant early and late apoptosis with diminished cell viability. Nano-immunoassay and metabolomics data on activation status of CB1R-mediated pro-apoptotic pathways found that ACPA inhibited Akt/PI3K pathway, glycolysis, TCA cycle, amino acid biosynthesis, and urea cycle and activated JNK pathway. ACPA lost its chemical stability after 24 hours tested by liquid chromatography-mass spectrometry (LC-MS/MS) assay. A novel ACPA-PCL nanoparticle system was developed by nanoprecipitation method and characterized. Sustained release of ACPA-PCL nanoparticles also reduced proliferation of NSCLC cells. Our results demonstrated that low dose ACPA and ACPA-PCL nanoparticle system harbor opportunities to be developed as a novel therapy in NSCLC patients that require further in vivo studies beforehand to validate its anticancer effect.

Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31 P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.

In 2008, the Food and Drug Administration of the US issued a warning about the neuropsychiatric side effects of montelukast. Previous clinical studies on montelukast have reported conflicting results and, to the best of our knowledge, no experimental studies concerning these side effects had been conducted. In the current study, the effect of montelukast on depression-like behavior in an ovalbumin (OVA)-induced mouse model was investigated. A total of 3 OVA challenges were applied at 2 week intervals for the persistence of asthma. Depression-like behavior was assessed using forced swim tests following each challenge and locomotor activities were evaluated using open field tests. At the end of the current study, plasma montelukast concentrations were measured and the development of asthma and effect of montelukast treatment were histopathologically examined. Inflammation scores that were increased in the OVA mice following all challenges were indicated to be reduced by montelukast treatment. The immobility time of mice increased beginning with the first challenge and this was also reduced by montelukast treatment. Montelukast administration to the control mice did not alter immobility times. Moreover, motor activity of the OVA and montelukast-treated mice were not altered. The results indicated there was no association between chronic montelukast treatment and depression. Furthermore, the chronic administration of montelukast to non-asthmatic mice did not increase immobility. However, depressive behavior increased at all time points in the OVA mice. These results indicated that chronic montelukast treatment is not associated with depression-like behavior and confirmed the association between asthma and depression. Further studies are required to provide an improved understanding of the neuropsychiatric side effects of montelukast., (Copyright: © Tel et al.)

Pronuciferine is a naturally occurring proaporphine alkaloid that belongs to isoquinoline alkaloids. The aim of this study is to investigate the neuroactivity of pronuciferine. We assessed the neuroprotective effect of pronuciferine against hydrogen peroxide (H 2 O 2 )-induced apoptosis in human neuronal SH-SY5Y cells. In addition, we measured the effect of pronuciferine on cell metabolites and brain-derived neurotrophic factor (BDNF) level in SH-SY5Y cells. In vitro result shows that pronuciferine at 10 µM significantly (P < .001) increased the proliferation of SH-SY5Y by 45%, and upon H 2 O 2 addition, pronuciferine significantly (P < .001) suppressed neuronal death caused by H 2 O 2 . Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics study revealed that pronuciferine has a high impact on glycine-serine-threonine pathway by changing the intracellular level of serine dimethylglycine, sarcosine, and threonine. Also, pronuciferine increased the intercellular level of aspartic acid, glutamine, and tryptophan. Additionally, pronuciferine significantly (P < .05) increased the intracellular BDNF protein expression at 10 µM. Therefore, pronuciferine is a neuroactive molecule that might act as a neuroprotective agent to prevent apoptosis in neurodegenerative diseases., (© 2020 Wiley Periodicals LLC.)

L-Carnitine has attracted much more attention especially in the treatment of crucial diseases such as diabetes, regional slimming, and obesity because of its metabolic activities. However, because of its short half-life, low bioavailability, and inability to be stored in the body, frequent dosing is required. In this study, L-carnitine-loaded liposome (lipo-carnitine) and PLGA nanoparticle (nano-carnitine) formulations were prepared and characterized. For lipo-carnitine and nano-carnitine formulations, particle size values were 97.88 ± 2.96 nm and 250.90 ± 6.15 nm; polydispersity index values were 0.35 ± 0.01 and 0.22 ± 0.03; zeta potential values were 6.36 ± 0.54 mV and - 32.80 ± 2.26 mV; and encapsulation efficiency percentage values were 14.26 ± 3.52% and 21.93 ± 4.17%, respectively. Comparative in vitro release studies of novel formulations and solution of L-carnitine revealed that L-carnitine released 90% of its content at the end of 1st hour. On the other hand, lipo-carnitine and nano-carnitine formulations maintained a controlled-release profile for 12 h. The in vitro efficacy of the formulations on cardiac fibroblasts (CFs) was evaluated by metabolomic studies and pathway analysis. Besides the prolonged release, lipo-carnitine/nano-carnitine formulations were also found to be effective on amino acid, carbohydrate, and lipid metabolisms. As a result, innovative nano-formulations were successfully developed as an alternative to conventional preparations which are available on the market.

Ethnopharmacological Relevance: Solanum melongena L. (eggplant) is used for treatment of rheumatism, beriberi, itching, toothache, bleeding, asthma, bronchitis, cholera, neuralgia and hemorrhoids in traditional medicine (Turkish, Chinese, and Indian). Hemorrhoids from these diseases, are common illness in all over the world, which are treated with various approaches including ethnobotanicals., Aim of the Study: This study aimed to evaluate the anti-hemorrhoidal activity of eggplant, an edible plant, which is commonly utilized around the world., Materials & Methods: In vivo anti-hemorrhoidal activity of the methanolic extract prepared from eggplant was evaluated by experimental hemorrhoid model, subsequently histological and biochemical analysis. Hemorrhoid, which was induced by applying croton oil to the anal area of the rats. Furthermore, the extract was screened for anti-inflammatory activity which is based on the inhibition of acetic acid-induced increase in capillary permeability. The healing potential was comparatively assessed with a reference Pilex® tablet and cream. Phytochemical analysis performed by HPLC. The amount of the major phenolic compound (chlorogenic acid) in extract was found by using HPLC method., Results: Histological and biochemical analysis demonstrated that eggplant extract is highly effective against hemorrhoid in comparison to the controls and the commercial preparation. In addition, the methanolic extract demonstrated significant inhibitory effect on acetic acid-induced increase in capillary permeability. The phytochemical studies identified major compound as chlorogenic acid (2.86%) by liquid chromatography., Conclusion: The eggplant calyxes, not edible, are easy to reach, by products/vast from the food sources. This is the first scientific evidence revealing that the eggplant extract has significant anti-hemorrhoidal and anti-inflammatory activity., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, metabolomics analyses have become increasingly common in the general scientific community as it is applied in several researches relating to diseases diagnosis. Identification and quantification of small molecules belonging to metabolism in biological systems have an important role in diagnosis of diseases. The combination of chromatography with mass spectrometry is used for the accurate and reproducible analysis of hundreds to thousands of metabolites in biological fluids or tissue samples. The number of metabolites that can be identified in biological fluids or tissue varies according to the gas (GC) or liquid (LC) chromatographic techniques used. The cover of these chromatographic techniques also differs from each other based on the metabolite group (polar, lipids, organic acid etc.). Consequently, some of the metabolites can only be analyzed using either GC or LC. However, more than one metabolite or metabolite group may be found altered in a particular disease. Thus, in order to find these alterations, metabolomics analyses that cover a wide range of metabolite groups are usually applied. In this regard, GC-MS and LC-MS techniques are mostly used together to identify completely all the altered metabolites during disease diagnosis. Using these combined techniques also allows identification of metabolite(s) with significantly altered phenotype. This review sheds light on metabolomics studies involving the simultaneous use of GC-MS and LC-MS. The review also discusses the coverage, sample preparation, data acquisition and data preprocessing for untargeted metabolomics studies. Moreover, the advantages and disadvantages of these methods were also evaluated. Finally, precautions and suggestions on how to perform metabolomics studies in an accurate, precise, complete and unbiased way were also outlined., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Down Syndrome is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21. Metabolomics is identification and quantification of small-molecule metabolites (molecular weight <1000 Da) in tissues, cells and physiological fluids within a certain period time. Metabolites are intermediate products of various types of biochemical reactions that participate in bonding metabolic pathways. In this study, metabolites such as 2-Hydroxybutyric acid, 3-Hydroxybutyric acid, β-Hydroxyisovaleric acid, Uracil, Glutamic acid, Maltose and Melezitose were chosen as the possible determinants/markers for the prenatal screening of Down Syndrome. Quantitative analysis of the metabolites conducted by GCMS method using 5 % phenyl / 95 % dimethylpolysiloxane (30 m ×0.25 mm, 0.25 μm film thickness) capillary column. The oven temperature was held constant at 60 °C for 1 min and ramped at 10 °C /min to 200 °C then ramped at 30 °C/min to 320 °C and hold for 6 min before cool-down, as helium mobile phase and flow rate of 2.8 mL/min and adding Myristic acid-d27 as an internal standard. Our method was validated by parameters of system suitability, stability, linearity, sensitivity, accuracy, precision, selectivity, robustness and ruggedness. The developed and validated method was applied to plasma samples taken from pregnant women with Down Syndrome (study group) and euploid fetuses (healthy group). The levels of these seven metabolites are statistically different (p < 0.05 for all) between the groups. It can be concluded that these relevant metabolites might be used for the prenatal screening of Down Syndrome., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Rivaroxaban (RXB) is a class II drug, according to the Biopharmaceutics Classification System. Since its bioavailability is low at high doses, dose proportionality is not achieved for pharmacokinetic parameters. However, when taken with food, its bioavailability increases at high doses. In this study, nanocrystal technology was used to increase the solubility and, hence, the bioavailability of RXB. Pluronic F127, pharmacoat 603, and PVP K-30 were used as stabilizers to prepare RXB nanosuspension, combining ball mill and high pressure homogenization methods. Particle sizes of RXB in nanosuspension (formulation A:348 nm; formulation B:403 nm) and nanocrystal formulations (formulation A:1167 nm; formulation B:606 nm) were significantly reduced (p < 0.05) compared to those of bulk RXB. In both formulations, 80% of the drug dissolved in 30 min. For dose proportionality evaluation, 3, 10, and 15 mg/kg of RXB nanosuspensions (formulation B) were administered to rabbits. The dose proportionality for AUC and C max of RXB nanocrystals was assessed by the power model, variance analysis of pharmacokinetic parameters, linear regression, and equivalence criterion methods. Dose proportionality for AUC was achieved at doses between 10-15 and 3-15 mg/kg. In conclusion, the preparation of a nanocrystal formulation of RXB improved its dissolution rate and pharmacokinetic profile.

Despite the new treatment strategies within the last 30 years, peripheral nerve injury (PNI) is still a worldwide clinical problem. The incidence rate of PNIs is 1 in 1000 individuals per year. In this study, we designed a composite nanoplatform for dual therapy in peripheral nerve injury and investigated the in-vivo efficacy in rat sciatic nerve crush injury model. Alpha-lipoic acid (ALA) was loaded into poly lactic-co-glycolic acid (PLGA) electrospun nanofibers which would release the drug in a faster manner and atorvastatin (ATR) loaded chitosan (CH) nanoparticles were embedded into PLGA nanofibers to provide sustained release. Sciatic nerve crush was generated via Yasargil aneurism clip with a holding force of 50 g/cm 2 . Nanofiber formulations were administered to the injured nerve immediately after trauma. Functional recovery of operated rat hind limb was evaluated using the sciatic functional index (SFI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and Basso, Beattie, and Bresnahan (BBB) test up to one month in the post-operative period at different time intervals. In addition to functional recovery assessments, ultrastructural and biochemical analyses were carried out on regenerated nerve fibers. L-929 mouse fibroblast cell line and B35 neuroblastoma cell line were used to investigate the cytotoxicity of nanofibers before in-vivo experiments. The neuroprotection potential of these novel nanocomposite fiber formulations has been demonstrated after local implantation of composite nanofiber sheets incorporating ALA and ATR, which contributed to the recovery of the motor and sensory function and nerve regeneration in a rat sciatic nerve crush injury model., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Identification of the low abundance of phytochemicals in plant extracts is very difficult. Pharmacological activity observed in such plants is not due to a single compound. In most cases, plant extracts show activity based on synergistic or antagonistic effects. Therefore, the idea of a holistic approach is more rational., Purpose: This study was planned to compare the metabolomics and proteomics profiles of Valeriana officinalis L. (Valerianaceae), Melissa officinalis L. (Lamiaceae), Hypericum perforatum L. (Hypericaceae) and Passiflora incarnata L. (Passifloraceae) used in sedative anxiolytic and sleep disorders. Integrated omics analyses were used to provide a better understanding of the effect of plant extracts on the brain-derived neurotrophic factor (BDNF) expression levels on the SH-SY5Y cell line by a holistic approach., Methods: Metabolomic profiling of the plants was performed using the GC-MS and LC-qTOF-MS systems, and the proteomics analysis using the LC-qTOF-MS system after trypsin digestion. The Human BDNF Quantikine ELISA kit was utilized to test BDNF expression activity on the SH-SY5Y cell line., Results: The investigated plant extracts showed a significant increase in BDNF expression (p < 0.05). M. officinalis was found as the most active extract. According to the correlation analyses between BDNF activity and metabolomics or proteomics level, 94 metabolites had a positive correlation while 23 metabolites had a highly negative correlation; those for proteins are 24 and 6, respectively., Conclusion: The multivariate data analysis revealed a similar metabolomics profile of H. perforatum and P. incarnata, which also had a similar activity profile. Remarkably, all the primary metabolites belonging to the Krebs Cycle (citric acid, fumaric acid, succinic acid, pyruvic acid, malic acid and citramalic acid, an analog of malic acid) were positively correlated with BDNF activity. Secondary metabolites with a high BDNF expression belonged to flavonoids, xanthone, coumarines, tannin, naphtalenes, terpenoids and carotenoid skeleton. Two proteins from the cytochrome P450 family (P450 71B11 and P450 94B3) were positively correlated with BDNF activity. Employing omics technologies in the plant research area will offer a better understanding of the role of plant extracts and may lead to the discovery of new compounds with specific activity., (Copyright © 2019. Published by Elsevier GmbH.)

The aim of this study was to demonstrate the altered metabolic infrastructure of pregnant women with methylenetetrahydrofolate reductase (MTHFR) polymorphisms at first trimester and during delivery. Eight singleton pregnant women with MTHFR polymorphisms were compared with 10 normal pregnant women. Maternal blood samples were obtained twice during their pregnancy period (between the 11th and 14th gestational weeks and during delivery). Metabolomic analysis was performed using GC-MS. The GC-MS based metabolomic profile helped identify 95 metabolites in the plasma samples. In the MTHFR group, the levels of 1-monohexadecanoylglycerol, pyrophosphate, benzoin, and linoleic acid significantly decreased (P ˂ 0.05 for all), whereas the levels of glyceric acid, l-tryptophan, l-alanine, l-proline, norvaline, l-threonine, and myo-inositol significantly increased (P ˂ 0.01 for the first two metabolites, P ˂ 0.05 for the others) at 11-14 gestational weeks. Conversely, the levels of benzoin, 1-monohexadecanoylglycerol, pyruvic acid, l-proline, phosphoric acid, epsilon-caprolactam, and pipecolic acid significantly decreased in the MTHFR group, whereas metabolites such as hexadecanoic acid and 2-hydroxybutyric acid increased significantly in the study group during delivery. An impaired energy metabolism pathway, vitamin B complex disorders, tendency for metabolic acidosis (oxidative stress), and the need for cell/tissue support seem prevalent in pregnancies with MTHFR polymorphisms., (© 2020 John Wiley & Sons, Ltd.)

A simple and reliable HPLC method was developed and validated for determination of rofecoxib in bovine serum albumin microsphere. The analyses were performed on a C18 column (150 x 4.6 mm, 5 μm particle size) at room temperature with UV detection at 272 nm. The mobile phase was composed of acetonitrile-0.1% o-phosphoric acid solution in water (1:1, v/v) mixture, and flow rate was set to 1 mL/min. The method was validated according to the international guidelines with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity, and robustness. The detection and quantification limit of the method were 1.0 μg/mL and 2.5 μg/mL, respectively. The method was linear in the range of 2.5-25 μg/mL with excellent determination coefficients (R2 >0.99). Intra-day and inter-day precision (<1.76% RSD) and accuracy (<0.55 % Bias) values of the method also fulfilled the required limits. It was concluded that the developed method was accurate, sensitive, precise, and reproducible according to the evaluation of the validation parameters. The applicability of the method was confirmed for in vitro quantification of rofecoxib in bovine serum albumin microspheres., Competing Interests: CONFLICT OF INTEREST: none declared, (Copyright © 2020 The Author(s).)

Cannabinoids and the endocannabinoid system significantly contributes to the airway inflammation. Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are two main enzymes responsible for the metabolism of the endocannabinoids anandamide (AEA) and 2-arachydonoyl glycerol (2-AG), respectively. In the present study, we aimed to investigate the effects of local and systemic FAAH and MAGL inhibitor treatments in experimental airway inflammation and tracheal hyperreactivity in mice. Airway inflammation was induced by intranasal (i.n.) lipopolysaccharide (LPS) application (60 μl; 0,1 mg/ml in PBS) to mice and the control group received PBS. Systemic (intraperitoneal (i.p.)) or local (i.n.) FAAH inhibitor URB597 and MAGL inhibitor JZL184 treatments were administered 1h before LPS/PBS application. Fourty 8 h after LPS/PBS application, tracheas were removed to assess airway reactivity, and the lungs and bronchoalveolar lavage (BAL) fluids were isolated for histopathological evaluation, cytokine and endocannabinoid measurements. LPS application lead to an increase in 5-hydroxytryptamine (5-HT) contractions in isolated tracheal rings while carbachol contractions remained unchanged. The increased 5-HT contractions were prevented by both systemic and local URB597 and JZL184 treatments. Systemic treatment with URB597 and JZL184, and local treatment with JZL184 reduced peribronchial and paranchymal inflammation in the LPS group while i.n. application of URB597 worsened the inflammation in the lungs. Systemic URB597 treatment increased lung AEA level whereas it had no effect on 2-AG level. However, JZL184 treatment increased 2-AG level by either systemic or local application, and also elevated AEA level. Inflammation-induced increase in neutrophil numbers was only prevented by systemic URB597 treatment. However, both URB597 and JZL184 treatments abolished the increased TNF-α level either they are administered systemically or locally. These results indicate that FAAH and MAGL inhibition may have a protective effect in airway inflammation and airway hyperreactivity, and therefore their therapeutic potential for airway diseases should be further investigated., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Exosomes are naturally secreted nano-vesicles consisting of biochemical molecules including RNAs, metabolites, lipids, and proteins, that emerge as diagnostic tools and disease-specific reporters. Here we offer a systematic and integrative approach for the simultaneous analysis of altered molecules namely metabolites, lipids, and proteins. These components tend to augment the discovery of low abundance signature components, and assist in explanation of molecular basis of colorectal cancer (CRC). In order to investigate CRC-derived exosomes, we selected mi-R19a, miR-21, miR-92a, and miR-1246 positive exosomes for downstream experiments. The overall multi-omic changes were investigated comparatively in cell culture and serum samples. Following a systematic multi-omic study, 37 (cell culture) and 31 (serum) metabolites; 130 (cell culture) and 56 (serum) lipids; 9 (cell culture) and 13 (serum) proteins were seen to be differentially expressed (p < 0.05), enabling discrimination between CRC and control. By using these enriched components, we demonstrated that the joint pathways mainly involving fatty acid and amino acid metabolism related pathways changed in CRC significantly. We conclude that this study increases our understanding of molecular basis of CRC, and provides potential exosomal biomarkers for the non-invasive detection, and discrimination of CRC., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Aim and Objective: Parkinson's disease (PD) is the second most common neurodegenerative disease. It is a multifactorial disorder (caused by aging, environmental, and genetic factors). Metabolomics can help explore the biomarker profiles for aging. Recent studies showed an association between the glutathione S-transferases (GSTs) polymorphisms and PD risk. The purpose of this study was to evaluate the association of this genetic polymorphism and the metabolomic profile in PD Tunisian patients, in order to identify effective biomarkers in the genetic differentiation., Materials and Methods: In this study, the metabolomic profile changes related to GSTs polymorphism were searched in 54 Tunisian PD patients treated with L-dopa, using a gas chromatography-mass spectrometry (GC-MS) technique., Results: The study results showed that mannose, methyl stearate, and three other unknown metabolites, increased in patients with GSTM1 positive genotype, while glycolic acid, porphine, monomethyl phosphate, fumaric acid, and three other unknown metabolites decreased in patients with GSTM1 positive genotype. Subsequently, the levels of glycolic acid, erythronic acid, lactic acid, citric acid, fructose, stearic acid, 2-amino-2-methyl-1,3-propanediol and three other unknown metabolites increased in patients with GSTM1 positive genotype, while the levels of proline, valine and two unknown metabolites decreased with GSTT1 positive genotype., Conclusion: All these altered metabolites are related to energy metabolism and it can be concluded that GSTs polymorphism based the shifting in energy metabolism and led to oxidative stress., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

In this study, we aimed to determine the interspecies interactions between Proteus mirabilis and Candida albicans. Mono and dual-species biofilms were grown in a microtiter plate and metabolomic analysis of the biofilms was performed. The effects of togetherness of two species on the expression levels of candidal virulence genes and urease and swarming activities of P.mirabilis were investigated. The growth of C.albicans was inhibited by P.mirabilis whereas the growth and swarming activity of P.mirabilis were increased by C.albicans. The inhibition of Candida cell growth was found to be biofilm specific. The alteration was not detected in urease activity. The expressions of EFG1, HWP1 and SAP2 genes were significantly down-regulated, however, LIP1 was upregulated by P.mirabilis. In the presence of P.mirabilis carbonhydrates, amino acids, polyamine and lipid metabolisms were altered in C.albicans. Interestingly, the putrescine level was increased up to 230 fold in dual-species biofilm compared to monospecies C.albicans biofilm. To our knowledge, this is the first study to investigate the impact of each microbial pathogen on the dual microbial environment by integration of metabolomic data., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

We aimed to configure impaired/altered metabolomic profiles of pregnant women carrying Down syndrome (DS) fetuses. The study involved 21 and 32 pregnant women with DS and euploid fetuses, respectively, as determined by prenatal screening and diagnosis as part of an antenatal care program. Metabolomic analyses were carried out using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) methods. A total of 95 metabolites were identified. GC-MS analysis indicated that levels of 2-hydroxybutyric acid, benzoic acid, nonanoic acid, 3-hydroxybutyric acid, and 2-ketoisocaproic acid were increased in the DS group, where beta-alanine, threonic acid, oxalic acid, alpha-tocopherol, uracil, 2-piperidone, and creatinine were decreased. However, LC-qTOF-MS analysis showed that lipid-related metabolites were decreased in women carrying DS fetuses, whereas creatine, N4-phosphoagmatine, citrate, 2,5-dioxopentanoate, 2-furoate, pyruvate, and fructose levels were increased. Pathway analysis was also performed using metabolites whose levels were significantly altered (p<0.05) between the groups, and the findings indicated that the biosynthesis pathways of aminoacyl-tRNA and "valine-leucine-isoleucine", and metabolism pathways of "glycine-serine-threonine", nitrogen, "alanine-aspartate-glutamate", propanoate, glycerophospholipid, cysteine, methionine, and phenylalanine were significantly altered. Our findings indicate a special type of metabolic status/syndrome in pregnant women with Down syndrome fetuses. It could be speculated that altered metabolic status might influence both gametogenesis and embryogenesis. Down syndrome is a complex genetic disorder that is important to detect prenatally, but may also be prevented by taking necessary precautions prior to pregnancy., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Alzheimer's Disease (AD) is one of the most challenging diseases faced by humankind. AD is still not classified as curable because of the complex structure of pathologies underlying it. As the mean life expectancy of the world population constantly increases, the prevalence of AD and treatment costs for AD also grow rapidly. Current state of the art for AD treatment mainly consists of palliative therapy aimed at providing symptomatic relief and improving the standard of living in patients with AD. However, different research groups are working on more effective and safe drug delivery options aimed at both symptomatic relief and treatment of the underlying mechanisms. In this review, the current prevalence of AD, health costs, pathologies, and available treatment options including the ones in the market and/or under trial have been reviewed. Data in the existing literature have been presented, and future opportunities have been discussed. It is our belief that these nanotechnological products provide the required efficacy and safety profiles to enable these formulations go through phase studies and enter the market after regulatory authority approval, as with cancer. Last, but not the least the metabolomic studies will be providing useful informative data on the early diagnosis of AD, thus may be clinical implications might be delayed with the administration of therapeutic agents at the initial state of the disease.