Fingerprinting Breast Milk; insights into Milk Exosomics.

Breast milk, often referred to as "liquid gold," is a complex biofluid that provides essential nutrients, immune factors, and developmental cues for newborns. Recent advancements in the field of exosome research have shed light on the critical role of exosomes in breast milk. Exosomes are nanosized vesicles that carry bioactive molecules, including proteins, lipids, nucleic acids, and miRNAs. These tiny messengers play a vital role in intercellular communication and are now being recognized as key players in infant health and development. This paper explores the emerging field of milk exosomics, emphasizing the potential of exosome fingerprinting to uncover valuable insights into the composition and function of breast milk. By deciphering the exosomal cargo, we can gain a deeper understanding of how breast milk influences neonatal health and may even pave the way for personalized nutrition strategies.

Metabolomics Point out the Effects of Carfilzomib on Aromatic Amino Acid Biosynthesis and Degradation.

(1) Carfilzomib (Cfz) is an antineoplastic agent indicated for the treatment of multiple myeloma. However, its beneficial action is attenuated by the occurrence of cardiotoxicity and nephrotoxicity as the most common adverse effects. Presently, there is well-established knowledge on the pathomechanisms related to these side effects; however, the research on the metabolic alterations provoked by the drug is limited. (2) An in vivo simulation of Cfz-induced toxicity was developed in (i) Cfz-treated and (ii) control mice. An RP-HRMS-based protocol and an advanced statistical treatment were used to investigate the impact of Cfz on the non-polar metabolome. (3) The differential analysis classified the Cfz-treated and control mice and resulted in a significant number of identified biomarkers with AUC > 0.9. The drug impaired the biosynthesis and degradation of aromatic amino acids (AAA) and led to alterations of uremic toxins in the renal and urine levels. Furthermore, the renal degradation of tryptophan was affected, inducing its degradation via the kynurenine pathway. (4) The renal levels of metabolites showed impaired excretion and degradation of AAAs. Cfz was, finally, correlated with the biosynthesis of renal dopamine, explaining the biochemical causes of water and ion retention and the increase in systolic pressure.

Prognostic Factors for Cardiotoxicity among Children with Cancer: Definition, Causes, and Diagnosis with Omics Technologies.

Improvements in the treatment of childhood cancer have considerably enhanced survival rates over the last decades to over 80% as of today. However, this great achievement has been accompanied by the occurrence of several early and long-term treatment-related complications major of which is cardiotoxicity. This article reviews the contemporary definition of cardiotoxicity, older and newer chemotherapeutic agents that are mainly involved in cardiotoxicity, routine process diagnoses, and methods using omics technology for early and preventive diagnosis. Chemotherapeutic agents and radiation therapies have been implicated as a cause of cardiotoxicity. In response, the area of cardio-oncology has developed into a crucial element of oncologic patient care, committed to the early diagnosis and treatment of adverse cardiac events. However, routine diagnosis and the monitoring of cardiotoxicity rely on electrocardiography and echocardiography. For the early detection of cardiotoxicity, in recent years, major studies have been conducted using biomarkers such as troponin, N-terminal pro b-natriuretic peptide, etc. Despite the refinements in diagnostics, severe limitations still exist due to the increase in the above-mentioned biomarkers only after significant cardiac damage has occurred. Lately, the research has expanded by introducing new technologies and finding new markers using the omics approach. These new markers could be used not only for early detection but also for the early prevention of cardiotoxicity. Omics science, which includes genomics, transcriptomics, proteomics, and metabolomics, offers new opportunities for biomarker discovery in cardiotoxicity and may provide an understanding of the mechanisms of cardiotoxicity beyond traditional technologies.

An Untargeted Metabolomics Approach on Carfilzomib-Induced Nephrotoxicity.

BACKGROUND: Carfilzomib (Cfz) is an anti-cancer drug related to cardiorenal adverse events, with cardiovascular and renal complications limiting its clinical use. Despite the important progress concerning the discovery of the underlying causes of Cfz-induced nephrotoxicity, the molecular/biochemical background is still not well clarified. Furthermore, the number of metabolomics-based studies concerning Cfz-induced nephrotoxicity is limited. METHODS: A metabolomics UPLC-HRMS-DIA methodology was applied to three bio-sample types i.e., plasma, kidney, and urine, obtained from two groups of mice, namely (i) Cfz (8 mg Cfz/ kg) and (ii) Control (0.9% NaCl) (n = 6 per group). Statistical analysis, involving univariate and multivariate tools, was applied for biomarker detection. Furthermore, a sub-study was developed, aiming to estimate metabolites' correlation among bio-samples, and to enlighten potential mechanisms. RESULTS: Cfz mostly affects the kidneys and urine metabolome. Fifty-four statistically important metabolites were discovered, and some of them have already been related to renal diseases. Furthermore, the correlations between bio-samples revealed patterns of metabolome alterations due to Cfz. CONCLUSIONS: Cfz causes metabolite retention in kidney and dysregulates (up and down) several metabolites associated with the occurrence of inflammation and oxidative stress.

Mineralocorticoid Receptor Pathway Is a Key Mediator of Carfilzomib-induced Nephrotoxicity: Preventive Role of Eplerenone.

Carfilzomib is an irreversible proteasome inhibitor indicated for relapsed/refractory multiple myeloma. Carfilzomib toxicity includes renal adverse effects (RAEs) of obscure pathobiology. Therefore, we investigated the mechanisms of nephrotoxicity developed by Carfilzomib. In a first experimental series, we used our previously established in vivo mouse models of Carfilzomib cardiotoxicity, that incorporated 2 and 4 doses of Carfilzomib, to identify whether Carfilzomib affects renal pathways. Hematology and biochemical analyses were performed, while kidneys underwent histological and molecular analyses. In a second and third experimental series, the 4 doses protocol was repeated for 24 hours urine collection and proteomic/metabolomic analyses. To test an experimental intervention, primary murine collecting duct tubular epithelial cells were treated with Carfilzomib and/or Eplerenone and Metformin. Finally, Eplerenone was orally co-administered with Carfilzomib daily (165 mg/kg) in the 4 doses protocol. We additionally used material from 7 patients to validate our findings and patients underwent biochemical analysis and assessment of renal mineralocorticoid receptor (MR) axis activation. In vivo screening showed that Carfilzomib-induced renal histological deficits and increased serum creatinine, urea, NGAL levels, and proteinuria only in the 4 doses protocol. Carfilzomib decreased diuresis, altered renal metabolism, and activated MR axis. This was consistent with the cytotoxicity found in primary murine collecting duct tubular epithelial cells, whereas Carfilzomib + Eplerenone co-administration abrogated Carfilzomib-related nephrotoxic effects in vitro and in vivo. Renal SGK-1, a marker of MR activation, increased in patients with Carfilzomib-related RAEs. Conclusively, Carfilzomib-induced renal MR/SGK-1 activation orchestrates RAEs and water retention both in vivo and in the clinical setting. MR blockade emerges as a potential therapeutic approach against Carfilzomib-related nephrotoxicity.

Chemometrically Assisted Optimization of Pregabalin Fluorescent Derivatization Reaction with a Novel Xanthone Analogue and Validation of the Method for the Determination of Pregabalin in Bulk via a Plate Reader.

Quantitation of chromophore-free analytes is always a challenge. To this purpose, derivatization of the analyte constitutes a common strategy, leading to a product with a strong signal. In the current study, a novel xanthone analogue was utilized for the first time for the derivatization of pregabalin, a model analyte with a primary amine moiety that lacks a chromophore. The fact that only the xanthene-based derivative, formed after the derivatization reaction fluoresces, enables avoiding its chromatographic separation from the reagent and thus reducing the analysis time of a series of samples in 1-2 min via a plate reader. The reaction conditions were optimized via a central composite design (CCD), with fluorescence signal as the measure of the yield. The following factors that affect the derivatization reaction were chosen: (a) temperature, (b) reaction time, and (c) triethylamine solution volume used to drive the reaction to completion. After the identification of the optimal conditions, the method was validated according to ICH guidelines, using a fluorescence plate reader for signal measurement (λex = 540, λem = 615 nm). Finally, the newly developed high-throughput method was applied to the determination of drug content in pregabalin bulk.

Exploring the metabolomic profile of cerebellum after exposure to acute stress.

Psychological stress and stress-related disorders constitute a major health problem in modern societies. Although the brain circuits involved in emotional processing are intensively studied, little is known about the implication of cerebellum in stress responses whereas the molecular changes induced by stress exposure in cerebellum remain largely unexplored. Here, we investigated the effects of acute stress exposure on mouse cerebellum. We used a forced swim test (FST) paradigm as an acute stressor. We then analyzed the cerebellar metabolomic profiles of stressed (n = 11) versus control (n = 11) male CD1 mice by a Nuclear Magnetic Resonance (NMR)-based, untargeted metabolomics approach. Our results showed altered levels of 19 out of the 47 annotated metabolites, which are implicated in neurotransmission and N-acetylaspartic acid (NAA) turnover, as well as in energy and purine/pyrimidine metabolism. We also correlated individual metabolite levels with FST behavioral parameters, and reported associations between FST readouts and levels of 4 metabolites. This work indicates an altered metabolomic signature after acute stress in the cerebellum and highlights a previously unexplored involvement of cerebellum in stress responses.

Phytochemical Differentiation of Saffron (Crocus sativus L.) by High Resolution Mass Spectrometry Metabolomic Studies.

The metabolite profiling of saffron (Crocus sativus L.) from several countries was measured by using ultra-performance liquid chromatography combined with high resolution mass spectrometry (UPLC-HR MS). Multivariate statistical analysis was employed to distinguish among the several samples of C. sativus L. from Greece, Italy, Morocco, Iran, India, Afghanistan and Kashmir. The results of this study showed that the phytochemical content in the samples of C. sativus L. were obviously diverse in the different countries of origin. The metabolomics approach was deemed to be the most suitable in order to evaluate the enormous array of putative metabolites among the saffron samples studied, and was able to provide a comparative phytochemical screening of these samples. Several markers have been identified that aided the differentiation of a group from its counterparts. This can be important for the selection of the appropriate saffron sample, in view of its health-promoting effect which occurs through the modulation of various biological and physiological processes.

Population Pharmacokinetics and Outcomes of Critically Ill Pediatric Patients Treated with Intravenous Colistin at Higher Than Recommended Doses.

Limited pharmacokinetic (PK) data suggest that currently recommended pediatric dosages of colistimethate sodium (CMS) by the Food and Drug Administration and European Medicines Agency may lead to suboptimal exposure, resulting in plasma colistin concentrations that are frequently <2 mg/liter. We conducted a population PK study in 17 critically ill patients 3 months to 13.75 years (median, 3.3 years) old who received CMS for infections caused by carbapenem-resistant Gram-negative bacteria. CMS was dosed at 200,000 IU/kg/day (6.6 mg colistin base activity [CBA]/kg/day; 6 patients), 300,000 IU/kg/day (9.9 mg CBA/kg/day; 10 patients), and 350,000 IU/kg/day (11.6 mg CBA/kg/day; 1 patient). Plasma colistin concentrations were determined using ultraperformance liquid chromatography combined with electrospray ionization-tandem mass spectrometry. Colistin PK was described by a one-compartment disposition model, including creatinine clearance, body weight, and the presence or absence of systemic inflammatory response syndrome (SIRS) as covariates (P < 0.05 for each). The average colistin plasma steady-state concentration (Css,avg) ranged from 1.11 to 8.47 mg/liter (median, 2.92 mg/liter). Ten patients had Css,avg of ≥2 mg/liter. The presence of SIRS was associated with decreased apparent clearance of colistin (47.8% of that without SIRS). The relationship between the number of milligrams of CBA per day needed to achieve each 1 mg/liter of plasma colistin Css,avg and creatinine clearance (in milliliters per minute) was described by linear regression with different slopes for patients with and without SIRS. Nephrotoxicity, probably unrelated to colistin, was observed in one patient. In conclusion, administration of CMS at the above doses improved exposure and was well tolerated. Apparent clearance of colistin was influenced by creatinine clearance and the presence or absence of SIRS.

Acute administration of the olive constituent, oleuropein, combined with ischemic postconditioning increases myocardial protection by modulating oxidative defense.

Oleuropein, one of the main polyphenolic constituents of olive, is cardioprotective against ischemia reperfusion injury (IRI). We aimed to assess the cardioprotection afforded by acute administration of oleuropein and to evaluate the underlying mechanism. Importantly, since antioxidant therapies have yielded inconclusive results in attenuating IRI-induced damage on top of conditioning strategies, we investigated whether oleuropein could enhance or imbed the cardioprotective manifestation of ischemic postconditioning (PostC). Oleuropein, given during ischemia as a single intravenous bolus dose reduced the infarct size compared to the control group both in rabbits and mice subjected to myocardial IRI. None of the inhibitors of the cardioprotective pathways, l-NAME, wortmannin and AG490, influence its infarct size limiting effects. Combined oleuropein and PostC cause further limitation of infarct size in comparison with PostC alone in both animal models. Oleuropein did not inhibit the calcium induced mitochondrial permeability transition pore opening in isolated mitochondria and did not increase cGMP production. To provide further insights to the different cardioprotective mechanism of oleuropein, we sought to characterize its anti-inflammatory potential in vivo. Oleuropein, PostC and their combination reduce inflammatory monocytes infiltration into the heart and the circulating monocyte cell population. Oleuropein's mechanism of action involves a direct protective effect on cardiomyocytes since it significantly increased their viability following simulated IRI as compared to non-treated cells. Οleuropein confers additive cardioprotection on top of PostC, via increasing the expression of the transcription factor Nrf-2 and its downstream targets in vivo. In conclusion, acute oleuropein administration during ischemia in combination with PostC provides robust and synergistic cardioprotection in experimental models of IRI by inducing antioxidant defense genes through Nrf-2 axis and independently of the classic cardioprotective signaling pathways (RISK, cGMP/PKG, SAFE).

Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein.

Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)-based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aß fibrillogenesis, a neuropathological hallmark of Alzheimer's Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aß1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aß1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aß1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

Synthesis, Biological Evaluation and Stability Studies of Some Novel Aza-Acridine Aminoderivatives.

Several new amino-substituted aza-acridine derivatives bearing a basic side chain have been designed and synthesized. The antiproliferative activity of the target compounds has been evaluated against three cancer cell lines-namely HCT-116 (colorectal), the uterine sarcoma MES-SA, and its doxorubicin-resistant variant MES-SA/Dx5. A limited number of the new acridines showed marginal cytotoxicity against the tested cell lines; nevertheless, these analogues possessed a similar substitution pattern. The moderate biological activity of these derivatives was attributed to their instability in aqueous media, which has been studied by mass spectrometry and computational chemistry experiments at the density functional level of theory (DFT).

Preliminary pharmacokinetic study of the anticancer 6BIO in mice using an UHPLC-MS/MS approach.

Indirubins represent a group of natural and synthetic products with bio-activities against numerous human cancer cell lines acting by inhibiting protein kinases. The natural sources of indirubins are plants of Isatis sp., Indigofera sp., and Polygonum sp., recombinant bacteria, mammalian urine and some marine mollusks. Specifically, the halogenated derivative 6-bromo indirubin-3'-oxime (6BIO) possesses increased selectivity against GSK-3. However, to our knowledge, no analytical method to determine 6BIO in biological fluids has been developed till now. Therefore, a rapid, sensitive and high throughput UHPLC-MS/MS methods were developed and validated to evaluate the concentrations of 6BIO in mice plasma. Plasma samples were pre-treated by protein precipation using cold mixture of methanol: acetonitrile (9:1, v/v) and separations were carried out on a Hypersil Gold C18 column (50 × 2.1 mm i.d.; 1.9 µm p.s.) using 0.1% acetic acid and methanol as mobile phase at a flow rate of 500 mL/min in a gradient mode. For quantitation, a hybrid LTQ-Orbitrap MS equipped with an electro-spray ionization source was used applying a selected reaction monitoring (SRM) option. The monitored transitions were m/z 354.0 → 324.0 for 6BIO and 297.1 → 282.1 for afromorsin (used as the internal standard) in the negative mode. Following the EMA, ICH and FDA guidelines for validation of analytical procedures, the assay method was fully validated in terms of selectivity, linearity, recovery, matrix effect, accuracy, precision, stability, and robustness. The validated methods were successfully applied to the pharmacokinetic studies of 6BIO following an oral administration to mice at the dose of 50 mg/kg. The results indicated that 6BIO possesses a Tmax of 30 min, a half-life of 1 h, and low plasma bioavailability.

Rapid isolation and characterization of crocins, picrocrocin, and crocetin from saffron using centrifugal partition chromatography and LC-MS.

This study demonstrates a simple method for one-step isolation of the main secondary metabolites of a hydroalcoholic extract of Crocus sativus stigmas (saffron) using step-gradient centrifugal partition chromatography. The analysis was performed in dual and elution-extrusion mode, using five biphasic systems of the solvents heptane/ethyl acetate/butanol/ethanol/water in ratios of 4:10:0:4:10, 1:13:0:4:10, 1:12:1:4:10, 1:10:3:4:10, and 1:7:6:4:10. Five major crocins, picrocrocin, and crocetin were directly isolated in one step. Scaling up to preparative level, allowed the recovery of significantly high quantities of pure compounds, especially trans-crocin-4, saffron's principal crocin. Comparing dual-mode and elution-extrusion, in dual-mode, the trans-crocin-4 containing fractions were co-eluted with a high amount of free ß-d-glucose. In contrast, absence of free ß-d-glucose was observed in the corresponding trans-crocin-4 fractions obtained by the second method denoting its superiority against dual-mode. Initiating analysis with the 4th solvent-system afforded selective isolation of trans-crocin-4, with reduction in experimental time and solvent consumption. Structure elucidation was performed by nuclear magnetic resonance spectroscopy, liquid chromatography with mass spectrometry, and high-resolution tandem mass spectrometry. The proposed methodology comprises an integrated approach for the purification and characterization of biologically active saffron components in a fast, selective, and environmentally friendly manner.

Pharmacokinetics of Daptomycin in Critically Ill Pediatric Patients.

The pharmacokinetics of daptomycin (10 mg/kg once daily) was studied in 4 critically ill pediatric patients aged 8 to 14 yrs. The area under the concentration-time curve from time zero to infinity (AUC0-∞) of plasma concentrations on day 1 ranged between 123.8 to 663.9 µg · h/ml, with lower values observed in septic and burn patients; clearance ranged from 15.1 to 80.7 ml/h/kg. Higher-than-recommended doses of daptomycin may be needed in septic children to ensure optimal drug exposure. Interpatient variability may suggest a role for therapeutic drug monitoring.

Hydroxytyrosol ameliorates metabolic, cardiovascular and liver changes in a rat model of diet-induced metabolic syndrome: Pharmacological and metabolism-based investigation.

Metabolic syndrome is a clustering of interrelated risk factors for cardiovascular disease and diabetes. The Mediterranean diet has been proposed as an important dietary pattern to confer cardioprotection by attenuating risk factors of metabolic syndrome. Hydroxytyrosol (HT) is present in olive fruit and oil, which are basic constituents of the Mediterranean diet. In this study, we have shown that treatment with HT (20mg/kg/d for 8 weeks) decreased adiposity, improved impaired glucose and insulin tolerance, improved endothelial function with lower systolic blood pressure, decreased left ventricular fibrosis and resultant diastolic stiffness and reduced markers of liver damage in a diet-induced rat model of metabolic syndrome. These results were accompanied by reduced infiltration of monocytes/macrophages into the heart with reduced biomarkers of oxidative stress. Furthermore, in an HRMS-based metabolism study of HT, we have identified 24 HT phase I and II metabolites, six of them being over-produced in high-starch, low-fat diet fed rats treated with HT compared to obese rats on high-carbohydrate, high-fat diet. These results provide direct evidence for cardioprotective effects of hydroxytyrosol by attenuation of metabolic risk factors. The implications of altered metabolism of HT in high-carbohydrate, high-fat diet fed obese rats warrant further investigation.

Synthesis and Pharmacological Evaluation of Novel Adenine-Hydrogen Sulfide Slow Release Hybrids Designed as Multitarget Cardioprotective Agents.

This work deals with the design, synthesis, and evaluation of the cardioprotective properties of a number of novel hybrid compounds combining the adenine nucleus with a suitable H2S slow-releasing moiety, coupled via a stable ether bond. The H2S release rate of the hybrids and their ability to increase cGMP were estimated in vitro. The most promising derivatives 4 and 11, both containing 4-hydroxythiobenzamide moiety as H2S donor, were selected for further in vivo evaluation. Their ability to release H2S in vivo was recorded using a new fully validated UPLC-DAD method. Both compounds reduced significantly the infarct size when administered at the end of sustained ischemia. Mechanistic studies showed that they conferred enhanced cardioprotection compared to adenine or 4-hydroxythiobenzamide. They activate the PKG/PLN pathway in the ischemic myocardium, suggesting that the combination of both pharmacophores results in synergistic cardioprotective activity through the combination of both molecular pathways that trigger cardioprotection.